CN1872333A - Placenta factor, preparation method and application - Google Patents

Abstract

The invention discloses a placenta factor as well as its preparation method and application. The placental factor can be prepared according to the following method: 1) Cut the placenta into pieces, perform tissue homogenization with a centrifugal force of 1770-2991.3 g, place the obtained homogenate in a water bath at 25-40° C. for 0.5-2.5 hours, and then incubate at 4 Centrifuge at 113.28-398.25g at -10°C for 20-40 minutes, and take the supernatant; 2) The supernatant obtained in step 1) is subjected to ultrafiltration with a molecular weight cut-off of 10KD, and the obtained ultrafiltration product is placenta factor. The method for preparing PF of the present invention has rich raw materials, simple preparation method and good stability. The placenta factor of the present invention is a small molecule polypeptide substance extracted from human placenta tissue, has few side effects, is safe to use, and has wide application prospects, and can be used for preventing and/or treating graft rejection in organ transplantation and hematopoietic stem cell transplantation and graft-versus-host disease (GVHD).

Description

A kind of placenta factor and its preparation method and application

technical field

The invention relates to a placenta factor and its preparation method and application.

Background technique

Graft-versus-host disease (GVHD) is a common complication after hematopoietic stem cell transplantation, and it is the main obstacle limiting the success of transplantation. Currently used immunosuppressants (eg, glucocorticoids, FK506, cyclosporin A, CellCept, etc.) and various methods of depleting T cells in the graft can reduce GVHD, but these methods increase the chance Incidence of infection, leukemia relapse, and transplant failure, accompanied by toxic side effects of the drug. Therefore, finding new ways to control GVHD has always been an important issue concerned by the field of hematology, and it is also one of the hot spots and difficulties in the study of transplantation immunology.

The fetus and the mother are different individuals but can coexist without immune rejection. This phenomenon suggests that there may be some components in the placenta that can make the fetus and mother produce immune tolerance. Domestic scholars used human placenta tissue as raw material to extract a small molecule polypeptide substance called placenta factor (PF) from the placenta. At present, there are mainly two methods for extracting PF. The first is the dialysis method: the placenta is shredded, homogenized at a high speed, after repeated freezing and thawing (-30°C to 40°C for 3 times), high-speed centrifugation (3000r/min, 25min), the supernatant is dialyzed for 48 hours, filtered to remove The second is the ultrafiltration method: cut the placenta into pieces, add 2 times normal saline, homogenate the tissue, centrifuge at a high speed (8000r/min, 20min), and take the supernatant for ultrafiltration to obtain PF. The physical and chemical properties research found that the appearance of PF is light yellow transparent liquid, and the qualitative reaction of protein is negative. Further biological activity studies found that PF can promote the recovery of sheep red blood cell receptors on the surface of T lymphocytes in vitro, promote the proliferation of T lymphocytes induced by PHA, and promote the secretion of IL-2 by lymphocytes and the expression of IL-2 receptors on the surface of lymphocytes. Expression; animal experiments also found that PF can enhance the phagocytosis of peritoneal macrophages, enhance the killing of tumor cells K562 by NK cells, and enhance the proliferation of T lymphocytes induced by PHA. At the same time, some scholars have used it to treat malignant tumors, viral hepatitis, bronchial asthma and allergic rhinitis and other diseases and have also obtained good curative effects.

Contents of the invention

The purpose of the present invention is to provide a placenta factor and its preparation method.

The placental factor provided by the present invention can be prepared according to the following method:

1) Shred the placenta, homogenate the tissue with a centrifugal force of 1770-2991.3g (10000-13000 rpm), incubate the obtained homogenate in a water bath at 25-40°C for 0.5-2.5 hours, then incubate at 4- Centrifuge at 13.28-398.25g (800-1500 rpm) at 10°C for 20-40 minutes, and take the supernatant;

2) The supernatant obtained in step 1) is subjected to ultrafiltration with a molecular weight cut-off of 10KD, and the obtained ultrafiltration product is placental factor.

The placental tissue can be homogenized in saline.

The centrifugal force of the tissue homogenate is preferably 2548.8g (12000 rpm).

The homogenate is preferably incubated in a water bath at 37°C for 1 hour.

After incubation, the homogenate was centrifuged at 113.28g (800 rpm) for 30 minutes at 4-10°C.

The placenta is human placenta.

In the above method, the step of sterilizing the placental factor obtained in step 2) is also included, for example, the ultrafiltration product is sterilized by filtration with a pore size of 220 μm.

The appearance of the PF prepared by the present invention is a light yellow transparent liquid, pH 6.5-7.5, and the qualitative protein is negative. The quality of the polypeptide measured by the Bradford method is 5.7-6.9mg/g fresh weight placenta, and the SDS-PAGE method measurement results show that the PF prepared by the present invention It mainly contains two components, the molecular weights are 9.187KD and 4.794KD respectively.

Another object of the present invention is to provide a drug for preventing and/or treating graft rejection and graft-versus-host disease.

The drug for preventing and/or treating graft rejection and graft-versus-host disease provided by the invention has the active ingredient of the placenta factor prepared by the invention.

When necessary, one or more pharmaceutically acceptable carriers can also be added to the above drugs. The carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrating agents, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field, and fragrances can also be added if necessary agents, sweeteners, etc.

The drugs for preventing and/or treating graft rejection and graft-versus-host disease can be introduced into the body by injection.

In the preparation method of the present invention, the step of incubation at 37° C. is added, and the centrifugation speed is reduced (from the previous high-speed centrifugation (such as 8000 rpm) to 800 rpm), which is different from the previous methods. The appearance of the PF of the present invention is light yellow transparent liquid, pH 6.5-7.5, protein qualitative is negative, and the quality of polypeptide determined by Bradford method is 5.7-6.9 mg/g fresh weight placenta, which is similar to previous literature reports. However, SDS-PAGE showed that the PF prepared by the present invention mainly contains two components with molecular weights of 9.187KD and 4.794KD, which is different from previous literature reports (the previously reported PF is a mixture of polypeptides with a molecular weight less than 5KD). This may be because incubation at 37°C and low-speed centrifugation are more conducive to retaining the active components with higher molecular weight in PF. In addition, through the research on its biological activity, it is also confirmed that the PF prepared by the present invention has a different biological activity than before. It has a strong immunosuppressive effect on T lymphocytes both in vivo and in vitro. After the application of PF in vivo, there is no obvious T lymphocyte depletion effect, but by reducing the proliferation and activation ability of T cells, inducing CD4 ⁺ CD25 ⁺ regulatory The production of T cells and CD8 ⁺ CD28 ^- suppressive T cells, regulation of secretion of Th1/Th2 cytokines and other ways to induce immune tolerance of the body, its effect is stronger than the traditional immunosuppressant cyclosporine A. At the same time, PF can increase the number of NK cells and NKT cells in vivo, and promote the killing of tumor cells by NK cells in vitro. Subsequently, in the allogeneic bone marrow transplantation model in mice, it was further confirmed that PF can prevent the occurrence of GVHD, reduce the severity of GVHD, and reduce the mortality of transplanted mice. In addition, the application of PF in vivo can also help the transplanted mouse donor hematopoiesis Implantation of cells and lymphocytes, and the above effects are significantly stronger than cyclosporine A. These results suggest that PF does not affect immune reconstitution after transplantation while inducing immune tolerance, and may preserve the body's resistance to pathogens and tumors by increasing the killing activity of NK cells and the number of NK cells and NKT cells At the same time, it can also promote the implantation of donor hematopoietic and lymphocytes, which will provide a new way to improve the effect of clinical organ transplantation and hematopoietic stem cell transplantation.

The method for preparing PF of the present invention has rich raw materials, simple preparation method and good stability. The placenta factor of the present invention is a small molecule polypeptide substance extracted from human placenta tissue, has few side effects, is safe to use, and has wide application prospects, and can be used for preventing and/or treating graft rejection in organ transplantation and hematopoietic stem cell transplantation and graft-versus-host disease (GVHD).

Description of drawings

Figure 1 is the SDS-PAGE electrophoresis result of PF

Figure 2 is the effect of PF on the proliferation of lymphocytes induced by PHA

Figure 3 shows the effect of PF on mixed lymphocyte reaction

Figure 4 shows the effect of PF on the expression of CD69 in T cells

Figure 5 shows the effect of PF on NK cells killing tumor cells

Figure 6 is the result of the effect of PF on the body weight of mice

Figure 7 shows the effect of PF on the peripheral blood leukocytes of mice

Figure 8 shows the effect of PF on the proliferation of mouse spleen lymphocytes induced by ConA and allogeneic antigens

Figure 9 shows the effect of PF on the number of T cells, CD4 ⁺ and CD8 ⁺ T cells and the ratio of CD4 ⁺ /CD8 ⁺ T cells

Figure 10 shows the effect of PF on the expression of CD28 in mouse spleen T cells and their subsets

Figure 11 shows the effect of PF on the number of CD4 ⁺ CD25 ⁺ regulatory T cells and CD8 ⁺ CD28 ^- suppressor T cells in mouse spleen

Figure 12 shows the effect of PF on the number of spleen NK cells and NKT cells in mice

Figure 13 is the effect of PF on IFN-γ and IL-4 in MLR culture supernatant

Figure 14 is the incidence of GVHD in each group of mice after bone marrow transplantation

Figure 15 is the survival curve of mice in each group after bone marrow transplantation

Figure 16A is the comparison of the occurrence time and number of cases of GVHD in three groups of mice

Figure 16B is the comparison of the death time and the number of dead cases of three groups of mice

Figure 17A is the change of body weight of mice in each group after bone marrow transplantation

Figure 17B is the change of peripheral blood leukocyte counts of mice in each group after bone marrow transplantation

Figure 18 shows the implantation of hematopoietic cells and lymphocytes from the three groups of mouse donors

Figure 19 is a 100-fold HE staining photo of the pathological changes in the liver and small intestine of the three groups of mice

Detailed ways

The experimental methods in the following examples are conventional methods unless otherwise specified.

Embodiment 1, the preparation of placenta factor (placenta factor, PF)

The material is healthy maternal placenta. Healthy puerperas were those who were tested negative for hepatitis B virus, hepatitis C virus, HIV, toxoplasma gondii and syphilis before delivery.

Remove the fascial blood vessels from the fresh placenta, wash it repeatedly with normal saline to completely remove the blood remaining in the interstitium, cut it into small pieces of 1×1cm2 ^size , add normal saline twice the volume of placental tissue for high-speed homogenization (12000 rpm, i.e. 2548.8g, 3 min/time, 10 times), the homogenate was incubated in a water bath at 37°C for 1 hour, then centrifuged at 4°C at a low speed (800 rpm, i.e. 113.28g) for 30 minutes, the The supernatant was placed in an AmiconUltra ultrafiltration tube (molecular weight cut-off 10KD) for centrifugal ultrafiltration. After ultrafiltration, the product was filtered and sterilized with a Millipore filter (220 μm) to obtain PF and stored at 4°C. And carry out the research of physical and chemical properties to the prepared PF, the results are as follows:

(1) Protein qualitative test

The protein qualitative test was carried out by heating acetic acid method and 20% sulfosalicylic acid method. The results showed that the appearance of PF was light yellow transparent liquid with pH 6.5-7.5. Heating acetic acid method and 20% sulfosalicylic acid method were negative for protein qualitative.

(2), determination of peptide quality (Bradford method)

Determination of peptide quality by Bradford method: Prepare protein standards of different concentrations, add Bradford staining solution, mix well, leave at room temperature for 2 minutes, and then measure the optical density value OD 590nm of the solution at a wavelength of _590nm . Draw the standard curve with the concentration of the protein sample as the ordinate and the optical density value as the abscissa. Take 500 microliters of the sample to be tested, dilute it to 1ml with distilled water, add Bradford dye solution, and measure OD _590nm according to the above method. And find out the concentration of the sample to be tested (that is, the mass concentration of the polypeptide) from the standard curve. Peptide mass (mg/g)=(peptide mass concentration×total volume of extract/sample volume during measurement)/placental tissue mass).

OD _590nm of the standard protein sample (bovine serum albumin, BSA) is plotted against the mass concentration of the standard protein sample to obtain a regression equation, peptide mass concentration (y)=-0.459+1.608OD _590nm (x), R ³ =0.847 , p=0.001, according to the OD _590nm of the protein sample to be tested, according to the regression equation, the peptide mass of PF is 5.70-6.86mg/g fresh weight placenta (6.28±0.58mg/g).

(3) Determination of the molecular weight of various components

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the molecular weight of the polypeptide. The separating gel, gap gel and stacking gel are respectively: 15.5% T (T represents the concentration of acrylamide) 6% C (C represents the degree of cross-linking of acrylamide) (containing 6mol/mL urea), 10% T 3% C and 4 %T 3%C. Ultra-low molecular weight protein peptide standard molecular weight range: 14.4KD-3.313KD.

The results of SDS-PAGE electrophoresis showed that PF was a mixture of small molecular polypeptides, which mainly contained two components (Figure 1). The migration distance of the ultra-low molecular weight protein polypeptide standard is plotted against the logarithm of the ultra-low molecular weight protein polypeptide standard molecular weight to obtain a regression equation, molecular weight logarithm (y)=4.286-0.269 (x) (migration distance), R ² =0.950 , p=0.025, according to the migration distance of the protein sample to be measured, the relative molecular weight of each component of PF obtained in the equation is 9.187KD and 4.794KD respectively; the molecular weight of α-thymosin is calculated as 3.158KD (theoretical value) through the regression equation is 3.108KD). In Fig. 1, 1. ultra-low molecular weight protein polypeptide standard (molecular weight range is 14.4KD to 3.313KD), 2. α-thymosin, 3. PF.

Embodiment 2, the biological activity of the PF prepared by the present invention

(1) In vitro biological activity research

1. Effect on PHA-induced lymphocyte proliferation

Human peripheral blood mononuclear cells (PBMC) were inoculated into a 96-well plate, and phytohemagglutinin (PHA) and different preparations were added at a final concentration of 10 μg/ml. The test was divided into three groups: different dilutions of PF (1/1, 1/2, 1/4, 1/6, 1/8 and 1/10 dilutions), different concentrations of cyclosporin A (cyclosporin A, CsA) (0.8, 0.6, 0.4, 0.2, 0.1 and 0.05 mg/ml) and normal saline (NS), and set up medium and control wells without PHA. Cultivate in 37°C 5% _CO2 incubator for 68 hours, use MTT method to detect, and calculate cell growth inhibition rate (growth inhibition rate (%)=1-(test well OD value/no stimulation well OD value)×100) .

The results are shown in Figure 2, showing that PF has a significant inhibitory effect on the proliferation of lymphocytes induced by PHA, in a dose-dependent manner, and as the dilution of PF increases, the inhibitory effect gradually weakens (compared P<0.0001 between each dose group and the NS group), Among them, the 1/1, 1/2 and 1/4 three dose groups compared with the CsA group P>0.05 (respectively 70.03% vs 67.91%, P=0.401; 67.80% vs 67.23%, P=0.744 and 59.33% vs 67.34 %, P=0.104). In Fig. 2, n=10, * indicates P<0.05 compared with NS group. The above results indicated that PF was comparable to CsA in inhibiting PHA-induced T cell proliferation in vitro.

2. Effect on mixed lymphocyte reaction:

PBMCs from different volunteers were used as responder cells and stimulator cells, and the stimulator cells were treated with mitomycin C (final concentration 60 μg/ml). The responder cells and stimulator cells were inoculated into 96-well plates at a ratio of 1:1, and different preparations were added. The grouping is the same as step 1, and set up control wells for purely reactive cells, purely stimulating cells, and purely medium. Cultivate for 6 days at 37° C. in a 5% CO ₂ incubator, detect with MTT method, and calculate the cell growth inhibition rate (same as step 1).

The results are shown in Figure 3, indicating that PF also has a significant inhibitory effect on the mixed lymphocyte reaction, also in a dose-dependent manner. With the increase of PF dilution, the inhibitory effect gradually weakens (P<0.0001 for each dose group compared with the NS group). Among them, the 1/1, 1/2, 1/4 and 1/6 four dose groups were compared with the CsA group with P>0.05 (respectively 78.90% vs 72.96%, P=0.708; 78.70% vs 73.43%, P=0.599; 73.04% vs 73.54%, P=1.000 and 66.02% vs 74.25%, P=0.469). In Fig. 3, n=10, * indicates P<0.05 compared with NS group. The above results indicated that the effect of PF on inhibiting mixed lymphocyte reaction in vitro was comparable to that of CsA.

3. Effect of phorbol ester (PMA) + ionomycin on the expression of T cell activation antigen CD69:

PBMC were inoculated into 24-well plates, and PMA (final concentration 200 ng/ml), ionomycin (final concentration 2 μg/ml) and different preparations were added to each well. The grouping is the same as step 1, and a negative control well is set up. Incubate in a 5% CO ₂ incubator at 37°C for 16-18 hours, and label antibodies according to the instructions (add anti-human CD3-FITC and isotype control-PE for the negative control; add anti-human CD3-FITC and anti-human CD69-PE for the rest of the samples) PE), the expression of CD69 in CD3 ⁺ cells was detected by flow cytometry. The expression of CD69 was represented by the percentage of CD69 ⁺ CD3 ⁺ cells in CD3 ⁺ cells.

The results are shown in Figure 4, indicating that PF has a significant down-regulation effect on the expression of T cell activation antigen CD69 after stimulation with PMA+ionomycin, in a dose-dependent manner. All group comparisons were P<0.05). 1/1, 1/2 and 1/4 of the three dose groups CD69 expression rate was lower than or equal to CsA group (respectively 19.58% vs 39.53%, P = 0.002; 35.53% vs 39.75%, P = 0.394 and 46.64% vs 39.70%, P=0.230). In Figure 4, n=8, * means P<0.05 compared with PF and NS group; # means P<0.05 compared with PF and CsA group. The above results indicated that both PF and CsA could inhibit T cell activation in vitro (PF vs CsA, P>0.05).

4. The effect on the killing activity of NK cells to kill tumor cells:

PBMC were inoculated into 24-well plates as effector cells, and different preparations were added. The test was divided into three groups: different dilutions of PF (1/1, 1/2, 1/4, 1/6, 1/8 and 1/10), different concentrations of CsA (0.2, 0.1 and 0.05mg/ml) And normal saline (NS). Incubate in a 5% CO ₂ incubator at 37°C for 12 hours, take out the effector cells, recount and inoculate the 96-well plate. The K562 cell line was inoculated into a 96-well plate as the target cells, and the effect-to-target ratio was 20:1. And set up effector cell spontaneous release well, target cell spontaneous release well, target cell maximum release well, volume correction release well and medium blank control well. The remaining steps were carried out according to the instructions, and the OD ₄₉₀ was detected by a microplate reader. And calculate the cell killing rate.

Killing rate (%)=(A-B-C)/(D-C)×100.

A = OD value of test well - OD value of blank control well

B = OD value of effector cell spontaneous release well - OD value of blank control well

C = OD value of target cell spontaneous release well - OD value of blank control well

D = OD value of target cell maximum release well - OD value of volume corrected well.

The results are shown in Figure 5, showing that the NK cell killing rates of the PF group were higher than or equal to the NS group, and the NK cell killing rates of the three dose groups of 1/4, 1/6 and 1/8 were higher than those of the NS group (32.32 %vs 15.23%, P=0.000; 22.73%vs15.23%, P=0.011 and 21.30% vs 15.23%, P=0.038); while 1/1, 1/2 and 1/10 three dose groups killed NK cells There was no significant difference in the rate between the NS group and the NS group (14.80% vs 15.23%, P=0.880; 16.48% vs 15.23%, P=0.663 and 18.96% vs 15.23%, P=0.199). The CsA group was significantly lower than the NS group (5.02% vs 15.23%, P=0.000; 6.69% vs 15.23%, P=0.004; 7.88% vs 15.23%, P=0.013). The above results indicated that, unlike CsA, the application of PF in vitro did not affect or even promote the killing of tumor cells by NK cells.

(2) In vivo biological activity research

Balb/c mice were randomly divided into three groups, 12 in each group. PF (20ml/kg.d), CsA (30mg/kg.d) and NS (0.4ml/mouse/day) were injected intraperitoneally, respectively, for 14 consecutive days, and the mice were sacrificed on the 15th day. The following bioactivity assays were performed:

1. Effect of PF on body weight and peripheral blood leukocyte count of mice

The body weight and peripheral blood white blood cell count of mice in each group were monitored every other day. The results of body weight changes are shown in Figure 6, indicating that the application of PF in vivo has no significant effect on the body weight of the mice, and the body weight of the mice in this group has been fluctuating at 20 g; however, the application of CsA in vivo caused the weight loss of the mice, decreased food intake, and decreased activity in the mice and mental deterioration. The results of changes in peripheral blood leukocytes are shown in Figure 7, indicating that the application of PF and CsA in vivo has no significant effect on the peripheral blood leukocyte counts of mice. In Fig. 6 and Fig. 7, n=12.

2. The effect of PF on the proliferation of mouse spleen lymphocytes induced by ConA:

The mice were sacrificed by dislocation of the cervical spine, the spleen was separated, and the spleen was ground in a glass homogenizer to make a spleen cell suspension, and the red blood cells were dissolved with hemolysin, and the concentration was adjusted to 1×10 ⁶ cells/ml for later use.

Spleen cells were inoculated into 96-well plates, and ConA was added at a final concentration of 4 μg/ml, and control wells with simple medium and no ConA were set up. 37° C. 5% CO ₂ incubator for 68 hours, MTT method detection, calculation of stimulation index (SI) (SI = test well OD value / NS group no stimulation well OD value).

The effect of PF on the proliferation of ConA-induced spleen lymphocytes in mice is shown in Figure 8. It shows that in the static state, the OD _570nm of mouse spleen lymphocytes in the PF group was lower than that in the NS group (0.384 vs 0.583, p=0.001) and the CsA group (0.384 vs 0.424, p=0.408). After ConA stimulation, the stimulation index of PF group was lower than that of NS group (0.890 vs 2.139, p=0.008) and CsA group (0.890 vs 1.312, p=0.423). The above results indicated that the effect of PF on inhibiting quiescence and ConA-induced proliferation of spleen lymphocytes in vivo was equivalent to that of CsA. In Fig. 8, n=12; cell proliferation activity in quiescent phase is represented by OD ₅₇₀ ; cell proliferation activity induced by ConA and allogeneic antigen is represented by stimulation index (SI); * indicates P<0.05 compared with NS group.

3. Effect of PF on mixed lymphocyte reactions (MLR):

Splenocytes from Balb/c mice (H-2 ^d ) and C57BL/6 mice (H-2 ^b ) were inoculated in a 96-well plate at a ratio of 1:1, and mixed well. Simultaneously set up simple reaction cells, simple stimulation cells and simple medium control wells. Cultivate in a 5% _CO2 incubator at 37°C for 6 days, detect with MTT method, and calculate the stimulation index (same as step 2). The results are shown in Figure 8, indicating that the stimulation index of the PF group was lower than that of the NS group (0.586vs 1.651, p=0.000) and the CsA group (0.586vs 0.608, p=0.801). The role of mouse spleen lymphocyte proliferation is comparable to that of CsA. In Fig. 8, n=12; cell proliferation activity in quiescent phase is represented by OD ₅₇₀ ; cell proliferation activity induced by ConA and allogeneic antigen is represented by stimulation index (SI); * indicates P<0.05 compared with NS group.

4. The effect of PF on T cells and their subsets, suppressor T cells (CD4 ⁺ CD25 ⁺ and CD8 ⁺ CD28 ^- T cells), natural killer (NK) cells and the number of natural killer T (NKT) cells, T cell surface Effects of co-stimulatory molecule CD28 expression:

Use fluorescently labeled specific antibodies to analyze splenocytes, take splenocytes, and each tube contains 2× ¹⁰⁶ cells/ml, and add CD3ε, CD4, CD8α, CD28, CD25, and Pan-NK 1.1 antibodies respectively according to the instructions , protected from light at 4°C for 30 minutes, resuspended cells, and tested on the machine, or fixed with 1% paraformaldehyde, stored at 4°C, and tested within 24 hours.

The effect of PF on the number of T cells, CD4 ⁺ and CD8 ⁺ T cells and the ratio of CD4 ⁺ /CD8 ⁺ T cells is shown in Figure 9, which shows that the numbers of T cells, CD4 ⁺ and CD8 ⁺ T cells in the PF group were significantly higher after treatment In CsA group (respectively 43.88vs31.78, P=0.004; 29.06vs 18.97, P=0.004; 8.71vs 5.96, P=0.026), but no difference with NS group (P>0.05). The ratio of CD4 ⁺ /CD8 ⁺ T cells in PF group was also higher than that in CsA and NS groups (but P>0.05). The results showed that, unlike CsA, PF had no T cell depleting effect in vivo, but induced immune tolerance by inhibiting the function of T cells. In Fig. 9, n=12; # indicates P<0.05 compared with CsA group.

The effect of PF on the expression of CD28 in T cells and their subsets is shown in Figure 10. After treatment, the expression of CD28 on the surface of T cells, CD4 ⁺ T and CD8 ⁺ T cells in the PF group was significantly lower than that in the CsA and NS groups (compared with CsA Group comparisons were: 20.29 vs 27.16, P = 0.006; 13.52 vs 18.58, P = 0.034; 4.67 vs 8.52, P = 0.024. Compared with NS group: 20.29 vs 30.46, P = 0.001; 13.52 vs 20.05, P=0.011; 4.67 vs 8.71, P=0.017). This result indicated that the application of PF in vivo could down-regulate the expression of co-stimulatory molecules in T cells and their subpopulations, thereby inducing T cell anergy, and the effect was stronger than that of CsA. In Fig. 10, n=12; the expression of CD28 is represented by the absolute number of CD28 ⁺ cells (×10 ⁶ cells/l); * indicates that the comparison between PF and NS group is P<0.05;# indicates that the comparison between PF and CsA group is P<0.05 .

The effect of PF on the number of CD8 ⁺ CD28 ^- suppressive T cells and CD4 ⁺ CD25 ⁺ regulatory T cells is shown in Figure 11, which shows that after administration, CD4 ⁺ D25 ⁺ regulatory T cells and CD8 ⁺ D28 ^- suppressor T The number of cells was significantly higher than that of CsA group and NS group (compared with NS group, CD4 ⁺ CD25 ⁺ T and CD8 ⁺ CD28 ^- T cell numbers were: 8.81vs4.95, P=0.002; 3.62vs 1.79, P =0.026), while the CsA group was lower than the NS group (respectively: 2.83vs4.95, P=0.052; 1.43vs 1.79, P=0.630). This result indicates that, unlike GsA, PF can induce immune tolerance in vivo by increasing CD8 ⁺ CD28 ^- suppressor T cells and CD4 ⁺ CD25 ⁺ regulatory T cells. In Fig. 11, n=12; * indicates P<0.05 compared with PF and NS group; # indicates P<0.05 compared with PF and CsA group.

The results of PF on the number of NK cells and NKT cells are shown in Figure 12, showing that the number of NK cells and NKT cells in the PF group was significantly higher than that in the NS and CsA groups after treatment (compared with the NS group, NK cells and NKT cells The numbers were: 4.8 vs 2.08, P=0.002; 1.96 vs 0.81, P=0.000). The number of NK cells in the CsA group was not significantly different from that in the NS group (2.32 vs 2.08, P=0.682), but the NKT cells in the CsA group were significantly lower than those in the NS group (0.41 vs 0.81, P=0.000). In Fig. 12, n=12; * indicates P<0.05 compared with PF and NS group; # indicates P<0.05 compared with PF and CsA group.

5. Determination of IL-4 and IFN-γ levels in MLR culture supernatant:

On the 6th day of MLR, the supernatant was collected by centrifugation, and the levels of IL-4 and IFN-γ were detected by BioSource mouse cytokine ELISA kit. The results are shown in Figure 13, indicating that the level of IFN-γ in the PF group was 1/4 of that in the NS group (42.33pg/ml vs 179.86pg/ml, P=0.000), and lower than that in the CsA group (42.33pg/ml vs 51 .61pg/ml, P=0.603); while the level of IL-4 was 2.5 times that of the NS group (110.8pg/ml vs 45.47pg/ml, P=0.006), and higher than that of the CsA group (110.8pg/ml vs 62.52pg /ml, P=0.031). The results show that the application of PF in vivo can significantly reduce the secretion of Th1 cytokines (such as IFN-γ), but promote the secretion of Th2 cytokines (such as IL-4), so that Th1 cells in vivo can shift to Th2 cells. In Fig. 13, n=12; * indicates P<0.05 compared with NS group; # indicates P<0.05 compared with CsA group.

(3) Effect of PF on graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT) in mice

The experimental method is as follows:

1. Preparation of recipient mice before transplantation

The mice were kept in the SPF animal room of the Experimental Animal Center of Peking University People's Hospital. The intestinal preparation was carried out by drinking an antibiotic solution containing gentamicin (320U/L) and erythromycin (250mg/L) 7 days before the transplantation, and lasted until 4 weeks after the transplantation. 4-6 hours before transplantation, the whole body was irradiated with ⁶⁰ Co-γ rays (irradiation room of Peking University Health Science Center). The radiation source was 150 cm away from the irradiation plane, the total irradiation dose was 8.0 Gy, the dose rate was 2.967 Gy/min, and the irradiation time was 2 minutes and 36 seconds.

2. Transplant group

The irradiated BaLB/C recipient mice were randomly divided into 4 groups A, B, C and D, with 22 rats in each group except group A, which had 7 rats. The mice in groups B, C and D were intraperitoneally injected with different preparations (PF, CsA and NS) from the day of transplantation for 14 consecutive days, and the injection doses were: PF (20ml/kg.d), CsA (30mg/kg.d) And NS (0.4ml/only/day).

Group A (simple irradiation group): no transplantation after irradiation.

Group B (PF transplantation group): Bone marrow and splenocytes of the donor mice were reinfused, and PF was continuously injected intraperitoneally for 14 days.

Group C (CsA transplantation group): Bone marrow and splenocytes of the donor mice were reinfused, and CsA was continuously injected intraperitoneally for 14 days.

Group D (NS control group): Bone marrow and splenocytes of donor mice were reinfused, and NS was continuously injected intraperitoneally for 14 days.

3. Isolation of donor mouse bone marrow cells and spleen cells

Donor mice were sacrificed by dismounting the cervical spine, and the tibia, femur and spleen of the mice were aseptically scissored. Cut off the metaphysis, and use a syringe filled with RPMI1640 solution to connect the No. 7 needle to flush the bone marrow cavity repeatedly until the bone marrow cavity turns white. Collect bone marrow cells and pass through the No. 4 needle to make a bone marrow cell suspension. Take the spleen of the donor mouse to make a spleen cell suspension (the method is the same as step 2). The above-mentioned bone marrow cells and spleen cells were stained with 4% trypan blue to detect cell viability ≥ 95%, and they were set aside.

4. Transplant

Except for the simple irradiation group, each recipient mouse in other groups was infused with 2×10 ⁸ cells/kg of bone marrow cells and 2×10 ⁸ cells/kg of spleen of donor mice through the tail vein 4-6 hours after irradiation. The mixed suspension of cells is 0.4-0.5ml. The ratio of bone marrow cells to spleen cells was 1:1.

5. Judgment criteria for survival and death

Death within 2 weeks after transplantation and WBC<0.5×10 ⁹ /L was defined as hematopoietic failure.

WBC>1.0×10 ⁹ /l, and fatigue, hair loss, hair raising, diarrhea, weight loss, arched back, and even death, the liver and small intestine of dead mice with GVHD pathological changes are GVHD.

Long-term survival is defined as survival greater than 60 days.

6. Observation indicators after mouse allo-BMT

(1) General situation:

Observe the weight, spirit, body shape, body position, hair, and diarrhea of the mice after transplantation. And the GVHD score of mice in each group was scored. Those with a score greater than 4 could be clinically diagnosed as GVHD, and those with a score greater than 6 were considered severe GVHD.

(2) Survival period and death:

Except for 7 mice in group A, 16 mice in each group were used for survival and mortality observation (up to 60 days after transplantation), and the remaining mice were tested for bone marrow cells and splenocyte chimerism.

(3) Hematopoietic function:

On the second day after the transplantation, 10 μl of blood was collected from the tails of the mice in each group, and added to the white blood cell counting solution diluted 10 times, and the recovery of white blood cells in the peripheral blood of each group was observed.

(4) Detection of bone marrow and spleen cell chimerism:

On the 14th and 21st days after transplantation, the recipient mice (3 in each group) were sacrificed, and the bone marrow and spleen were collected to make bone marrow cell and spleen cell suspensions respectively. Flow cytometry was used to detect H-2D ^b in bone marrow cells and spleen cells. The percentage of positive cells can reflect the engraftment of hematopoietic cells and lymphocytes in recipient mice.

(5) Pathological form of GVHD:

Each group was observed for 60 days. Liver and intestinal tissues were taken from mice with GVHD before they died, fixed with 10% formaldehyde solution, embedded in paraffin, sectioned, HE stained, and observed under a light microscope. Liver and intestinal tissues were taken from long-term surviving mice for pathological observation before sacrifice.

The experimental results are as follows:

1. The observed results of the survival period, mortality and GVHD occurrence of mice after transplantation showed that 7 mice in group A began to die on the 4th day after irradiation, and all died within 12 days, all of which died of hematopoietic failure. The survival time (8.143± 1.056) days.

Eleven mice in group D began to show symptoms such as fatigue, significantly decreased activity, less food, shrugging hair, diarrhea, weight loss, and severe arched back on the 4th day after transplantation (GVHD score was 7.18±1.33), and the remaining 5 mice The above symptoms appeared on the 6th day after transplantation. The median onset time of GVHD was the 4th day after transplantation, and the incidence rate of GVHD was 100% 14 days after transplantation (Fig. 14 and Table 1). The mice in group D began to die on the 5th day after transplantation, the median time of death was 8.5 days, all died within 19 days, and the survival time was (10.00±1.268) days (Figure 15 and Table 2).

The above symptoms began to appear on the 6th day after transplantation in group C mice (GVHD score was 6.07±0.83 points). The median onset time of GVHD was 6 days, and the incidence rate of GVHD was 100% 14 days after transplantation (see Figure 14 and Table 1). The mice in group C began to die on the 8th day after transplantation, the median time of death was 14.5 days, all died within 25 days, and the survival time was (14.938±1.453) days, which was longer than that of group D mice (14.94 vs 10.00, P=0.015) , but significantly shorter than that of group B mice (14.94 vs 48.15, P=0.000) (see Figure 15 and Table 2).

Five of the 16 mice in group B showed slight activity reduction, weight loss, and shrugging (GVHD score was 4 points) on the 8th day after transplantation, and 2 of them died of GVHD within 29 days (GVHD score was 6 at the time of death). points, manifested as weight loss, slight decrease in activity, arched back and hair shrugging at rest), and the other 3 survived during the observation period, and as time went by, the above symptoms gradually disappeared. Another mouse developed the above symptoms on day 26 after transplantation (GVHD score was 4 points), accompanied by mild diarrhea, and died on day 31 after transplantation (GVHD score was 5 points at the time of death). After 30 days, the remaining 4 mice showed mild abdominal hair loss, weight loss, slight activity reduction, bowed back and hair shrugging at rest, and died on the 47th, 58th, 43rd and 40th day after transplantation respectively (at the time of death GVHD score was 4.75±1.26 points). The median onset time of GVHD in group B mice was 46 days, which was significantly later than that in groups C and D (P<0.0001). At the same time, the incidence of GVHD in group B mice was also significantly lower than that in groups C and D (P<0.0001). The incidence of GVHD was 0 at 14 days after transplantation, 18.7% at 30 days, 39.1% at 45 days, and 49.2% at 60 days (See Table 1 and Figure 14). A total of 14/16 mice survived longer than 30 days. The survival period of 6/9 mice was greater than 45 days, and the survival period of 2/5 mice was greater than 60 days. Using Kaplan-Meier to evaluate the survival curves of the four groups of mice, it can be seen that the survival time of the mice in group B was significantly longer than that of other groups, and the average survival time was (48.145±3.382) days. Compared with other groups in pairs (A, C, D group) the difference was significant (P<0.0001). The 30-day, 45-day and 60-day survival rates of mice in group B were significantly higher than those in groups C and D (P<0.0001). The 30-day survival rates were 87.5%, 0%, and 0%, respectively, the 45-day survival rates were 60.9%, 0%, and 0%, and the 60-day survival rates were 30.5%, 0%, and 0% (see Table 2 and Table 2). Figure 15).

The GVHD score of group B was significantly lower than that of group D (P<0.0001). Among them, 7 days after transplantation, it was 2.375 vs 7.00, P=0.000; 14 days after transplantation, it was 3.125 vs 8.111, P=0.000; 21 days after transplantation, it was 1.5 vs 8.5, P=0.000. And the GVHD score of group B was also significantly lower than that of group C (P<0.01), respectively: 2.375 vs 6.31 at 7 days after transplantation, P=0.000; 3.125 vs 7.38 at 14 days after transplantation, P=0.000; 21 days after transplantation 1.5 vs 7.86, P＝0.000; 30 days after transplantation, 1.875 vs 8.5, P＝0.001. The GVHD score of group C was lower than that of group D, but the difference was not statistically significant (see Table 3).

Figure 16A shows the time and number of cases of GVHD clinical manifestations in the three groups of mice. It can be seen that the time of GVHD symptoms in group B mice was significantly later than that in groups C and D, and the time of GVHD in mice in group C was the same as that in group D. There was no significant difference between groups (the median onset time of GVHD in the three groups were: 46 days vs 6 days vs 4 days); and the number of cases of GVHD in group B mice was significantly less than that in groups C and D (respectively: 7 vs 16 vs 16). Figure 16B shows the time of death and the number of death cases in the three groups of mice. It can be seen from the figure that the death time of the mice in group B was significantly later than that in groups C and D, while the death time of mice in group C was not significantly different from that in group D (three The median death time of mice in group B was 40 days vs 14.5 days vs 8.5 days), and the number of dead mice in group B was significantly less than that in groups C and D (respectively: 7 vs 16 vs 16). In FIG. 16A and FIG. 16B, n=16.

The above results suggest that the application of PF in vivo can significantly reduce the occurrence of acute GVHD and GVHD-related death in allogeneic bone marrow transplantation mice, reduce the severity of GVHD in recipient mice, prolong the survival period of recipient mice, and improve the overall survival rate. And the above effects are stronger than CsA (P<0.01).

In Fig. 14, n=16; * represent and compare P value with B group (PF transplantation group) and D group (NS transplantation group); # represent B group and C group (CsA transplantation group) compare P value; & represent C group P value compared with group D. Group A in Fig. 15 has 7 mice; each of the other groups has 16 mice. * indicates the P value compared with group B (PF transplantation group) and D group (NS transplantation group); # indicates the P value compared with group B and C (CsA transplantation group); & indicates the P value compared with group C and D group.

Table 1. Comparison of the occurrence of GVHD in each group of mice after Allo-BMT (n=16) group Median time (d) 14d(%) 30d(%) 45d(%) 60d(%) Group B Group C Group D 46 ^* #64 0 ^* #100100 18.7 ^* #100100 39.1 ^* # 49.2 ^* #

Note: ^* indicates P<0.01 compared with group B (PF transplantation group) and D group (NS transplantation group), # indicates P<0.01 compared with group B and C (CsA transplantation group)

Table 2. Comparison of survival of mice in each group after Allo-BMT group no survival time 30d Survival % 45d Survival % 60d survival rate% minimum number of days Maximum number of days x±s Group A, Group B, Group C, Group D 7161616 42784 1260+2519 8.14±1.0648.15±3.38 ^* #14.94±1.4510.00±1.27 087.5 ^* #00 060.9 ^* #00 030.5 ^* #00

Note: ^* indicates P<0.01 compared with group B (PF transplantation group) and D group (NS transplantation group), # indicates P<0.01 compared with group B and C (CsA transplantation group).

Table 3. Comparison of GVHD scores of mice in each group after Allo-BMT (n=16) group 7d 14d 21d 30d 45d 60d Group B Group C Group D 2.38±0.81 ^* #6.31±1.017.00±1.55 3.13±0.72 ^* #7.38±1.198.11±0.93 1.5±0.89 ^* #7.86±0.908.5±0.58 1.88±0.13#8.5±0.71 2.5±0.20# 1.33±0.16#

Note: ^* indicates P<0.01 compared with group B (PF transplantation group) and D group (NS transplantation group), # indicates P<0.01 compared with group B and C (CsA transplantation group)

2. The body weight changes and white blood cell count results of the mice in each group after transplantation are shown in Figure 17A, Figure 17B and Table 4, indicating that the weight of the mice in group A continued to decrease after transplantation, and the peripheral blood white blood cells were <0.5×10 ⁹ /L, 1 They died one after another around the week.

The weight of the mice in group B began to decrease 2 days after the transplantation, from an average of 20.11 g to 15.74 g, and gradually recovered to 20.7 g from the 10th day. Peripheral blood leukocytes began to decrease on the second day, reached the lowest point of 0.3×10 ⁹ /L on the fourth day, and then gradually returned to normal. From the 6th day after transplantation, the peripheral blood leukocyte counts of mice in group B were higher than those in groups C and D (P<0.05). This result suggested that the application of PF in vivo could promote the recovery of hematopoiesis in mice after transplantation.

The weight of the mice in group C began to decrease on the second day after transplantation, and reached the lowest level on the 14th day (from an average of 20.14 g to 14.89 g), and then gradually recovered to about 15 g. The white blood cell count began to decrease on the second day, reached the lowest point of 0.35×10 ⁹ /L on the fourth day, and then gradually returned to normal. From the 6th day after transplantation, the peripheral blood leukocyte counts of mice in group C were higher than those in group D (day 6: 2.06 vs 1.39, P=0.01; day 8: 5.42 vs 3.34, P=0.054; day 14: 10.13 vs 3.63, P=0.042).

The body weight of the mice in group D began to decrease on the second day after transplantation, reached the lowest point on the sixth day from an average of 19.95 g to 14.47 g, and then gradually increased to about 17 g. Peripheral blood leukocytes began to decrease on the second day, reached the lowest point of 0.3×10 ⁹ /L on the fourth day, and then gradually returned to normal levels. In Fig. 17A, Fig. 17B and Table 4, n=16, * represents P<0.05 compared with group B (PF transplantation group) and D group (NS transplantation group), # represents P<0.05 compared with group B and C group (CsA transplantation group) 0.05.

Table 4. Comparison of peripheral blood white blood cell counts of mice in each group after transplantation (n=16). time (d) Group B Group C Group D Group A Compare B with C Compare B with D C vs. D 0246814202842 11.52±3.103.9±3.12041±0.112.98±0.58 ^* #7.93±3.21 ^* #16.38±4.57 ^* #16.68±4.57#16.43±4.0716.61±4.07 9.72±2.752.9±2.180.36±0.122.06±0.635.42±1.7210.13±3.7213.05±0.39 1053±3.54.08±4.540.32±0.11.39±0.573.34±1.233.63±0.54 10.39±4.173±0.930.44±0.240.22±0.130.1±0 0.1110.4070.1880.0000.0080.0060.007 0.2890.8900.0280.0000.0000.000 0.5830.3340.3460.0100.0540.042

Note: ^* means P<0.05 compared with group B (PF transplantation group) and D group (NS transplantation group), # means P<0.05 compared with group B (PF transplantation group)

3. Donor hematopoietic cell implantation

The ratio of H-2D ^{b +} cells in bone marrow and spleen cells was detected with PE-H-2D b monoclonal antibody at 14 and 21 days after transplantation, respectively. The experimental results are shown in A and B in Figure 18. On the 14th day after transplantation, the proportion of H-2D ^b+ cells in the mouse bone marrow cells of group B was significantly higher than that of groups C and D (P=0.000) (respectively: 91.55% vs 68.9% vs 65.13%), compared group C and D, P=0.004; and the proportion of H-2D ^b+ cells in bone marrow cells of mice in group B on day 21 after transplantation was not different from that in group C (99.08% vs 98.6%, P=0.379). At the same time, the experiment also found that the proportion of H-2D ^b+ cells in spleen lymphocytes of mice in group B was higher than that in group D (P=0.001) on the 14th day after transplantation, but there was no significant difference compared with group C (P=0.147). They were 97.7% vs 96.94% vs 94.86%, respectively; and the proportion of H-2D ^b+ cells in the splenic lymphocytes of mice in group B was not significantly different from that in group C on day 21 after transplantation (98.02% vs 98.49%, P=0.507 ). The above results suggested that the hematopoietic cells and lymphocytes of the donor mice were successfully engrafted in the recipient mice on the 21st day after transplantation, and the application of PF in vivo could significantly promote the engraftment of the donor hematopoietic cells and lymphocytes. In FIG. 18, n=3, and the engraftment status of donors is represented by the percentage of H-2D ^b+ cells. * indicates P<0.01 compared with group B (PF transplantation group) and D group (NS transplantation group); # indicates P<0.01 compared with group B (PF transplantation group) and C group (CsA transplantation group). (Note: Since all the mice in group D died on day 21 after transplantation, the implantation of mice in group D could not be detected).

4. Pathological changes

The mice in group D all had clinical manifestations of GVHD. Pathological examination showed atrophy of multiple organs, black color, obvious atrophy of liver and spleen, and spot-like hemorrhages on the surface of the liver. Pathological section showed swelling and degeneration of liver cells, disorder of liver cell cords, obvious expansion and congestion of portal vein and central vein, massive lymphocyte infiltration in hepatic sinusoid and central portal area, apoptosis of intrahepatic bile duct epithelial cells, and massive lymphocyte infiltration ( Fig. 19 middle a): Intestinal villi necrosis and falling off, crypt structure destruction and disappearance, many apoptotic bodies and necrotic cell fragments can be seen in crypts, mucosa and submucosa are obviously edematous and congested, submucosa has a large number of lymphocyte infiltration (Fig. 19 Middle b). The pathological changes in the liver and small intestine of the mice in this group were consistent, which were consistent with the diagnosis of GVHD.

The pathological changes of mice in group C were the same as those in group D.

The pathological changes of mice in group B were only mild inflammatory reaction: mild degeneration of liver cells (c) in Fig. 19 . The small intestinal mucosa was intact, with mild submucosal edema, and a small amount of lymphocyte infiltration in the small intestine (Figure 19 d).

Pathological examination indicated that PF could alleviate the liver and intestinal damage caused by GVHD.

In summary, biological activity studies confirmed that PF has a strong immunosuppressive effect on T lymphocytes in vitro and in vivo. After in vivo application of PF, there is no obvious T lymphocyte depletion effect, but by reducing the proliferation and activation ability of T cells, inducing the production of CD4 ⁺ CD25 ⁺ regulatory T cells and CD8 ⁺ CD28 ^- suppressor T cells, regulating Th1 / Th2 cytokine secretion and other ways to induce the body immune tolerance, its effect is stronger than the traditional immunosuppressant cyclosporine A. At the same time, PF can increase the number of NK cells and NKT cells in vivo, and promote the killing of tumor cells by NK cells in vitro. Subsequently, in the allogeneic bone marrow transplantation model in mice, it was further confirmed that PF can prevent the occurrence of GVHD, reduce the severity of GVHD, and reduce the mortality of transplanted mice. In addition, the application of PF in vivo can also help the transplanted mouse donor hematopoiesis Implantation of cells and lymphocytes, and the above effects are significantly stronger than cyclosporine A. These results suggest that PF may not affect immune reconstitution after transplantation while inducing immune tolerance, and may preserve the body's resistance to pathogens and tumors by increasing the killing activity of NK cells and the number of NK cells and NKT cells At the same time, it can also promote the implantation of donor hematopoietic and lymphocytes, which will provide a new way to improve the effect of clinical organ transplantation and hematopoietic stem cell transplantation.

Claims (10)

1. A method for preparing placental factor, comprising the following steps: 1) Shred the placenta, perform tissue homogenization with a centrifugal force of 1770-2991.3g, incubate the obtained homogenate in a water bath at 25-40°C for 0.5-2.5 hours, then centrifuge at 113.28-398.25g at 4-10°C for 20 ~40 minutes, take the supernatant; 2) The supernatant obtained in step 1) is subjected to ultrafiltration with a molecular weight cut-off of 10KD, and the obtained ultrafiltration product is placental factor. 2. The method according to claim 1, characterized in that: the centrifugal force of the tissue homogenate is 2548.8g. 3. The method according to claim 1 or 2, characterized in that: the homogenate is incubated in a water bath at 37°C for 1 hour. 4. The method according to claim 1 or 2, characterized in that: after incubation, the homogenate is centrifuged at 113.28g for 30 minutes at 4-10°C. 5. The method of claim 1, wherein the placenta is human placenta. 6. The method according to claim 1, further comprising the step of sterilizing the placental factors obtained in step 2). 7. Placental factors produced by the method of any one of claims 1 to 6. 8. The placental factor according to claim 7, characterized in that: said placental factor mainly contains two polypeptides with molecular weights of 9.187KD and 4.794KD respectively. 9. A drug for preventing and/or treating graft rejection and graft-versus-host disease, the active ingredient of which is the placenta factor as claimed in claim 7 or 8. 10. The medicament according to claim 9, characterized in that: the dosage form of the medicament is injection or powder injection.

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