CN1306028C - In vitro purification of self-sanguifacient dry cell and kit - Google Patents

Abstract

The present invention provides a method for purifying an auto-hemopoietic stem cell in vitro. The present invention is characterized in that the method comprises the steps that a hemopoietic stem cell containing a tumor cell is in contact with a proliferated gonad virus; the proliferated gonad virus enters the tumor cell. The present invention also provides a kit for the method for purifying the auto-hemopoietic stem cell in vitro.

Description

Method and kit for in vitro purification of autologous hematopoietic stem cells

field of invention

The invention relates to the field of biomedicine, in particular to an in vitro purification method for tumor cells mixed in hematopoietic stem cells during autologous hematopoietic stem cell transplantation, a corresponding kit and a treatment method for tumors.

Background of the invention

High-dose chemoradiotherapy combined with hematopoietic stem cell transplantation is an important technique for the treatment of malignant tumors. According to the source of hematopoietic stem cells, hematopoietic stem cell transplantation can be divided into autologous hematopoietic stem cell transplantation and allogeneic hematopoietic stem cell transplantation. Compared with allogeneic transplantation, autologous hematopoietic stem cell transplantation has the advantages of not being limited by donors, low transplant-related mortality, high safety, and low cost. At present, autologous hematopoietic stem cell transplantation has been widely used in the treatment of malignant tumors, and it has a positive effect on patients with multiple myeloma and malignant lymphoma (KumarA, et al., Lancet Oncol.2003; 4: 293-304; Schouten HC , et al., J. Clin Oncol. 2003; 21(21): 3918-27). Over the past 10 years, with the improvement of peripheral blood hematopoietic stem cell transplantation technology, this technology has been used in refractory breast cancer, testicular cancer, ovarian cancer, small cell lung cancer, nasopharyngeal cancer, childhood fibroblastoma, In the research on the treatment of solid tumors such as soft tissue sarcoma, many data have shown good preliminary curative effect.

Although autologous hematopoietic stem cell transplantation has many advantages, the reinfusion of tumor cells that may be brought about by autologous hematopoietic stem cell transplantation is often the source of tumor recurrence (Brenner MK, et al., Lancet.1993; 341:85-68), especially For hematological tumors such as multiple myeloma, as well as tumors with a high rate of bone marrow invasion such as intermediate-to-high-grade lymphoma, breast cancer, childhood neuroblastoma, and small-cell lung cancer (Armitage JO, Antman K. High-Dose Cancer Therapy: Pharmacology, Hematopoietins, Stem cells (Third edition). 2000 by LIPPINCOTT WILLIAMS & WILKINS, pp301-331).

In the early 1990s, based on the difference between the CD34 antigen on the surface of hematopoietic stem cells and malignant tumor cells, someone developed the CD34-positive cell immunomagnetic bead screening and purification technology (MACS), which once brought great hope to the purification of autologous hematopoietic stem cells in vitro, but After more than 10 years of clinical evaluation, it was found that although this technology can reduce the number of tumor cells by 2 to 6 logarithmic levels, the remaining tumor cells can still lead to tumor recurrence.

Then, according to the natural difference of adenovirus receptors on the surface of hematopoietic stem cells and malignant tumor cells, a method of purifying hematopoietic stem cells by using replication-defective adenoviruses carrying tumor suppressor genes or suicide genes was proposed. However, because the replication-deficient adenovirus used in this method is only a carrier, it cannot kill tumor cells by itself, and its killing of tumor cells mainly depends on the foreign genes carried, while the anti-tumor effect of foreign genes depends on Whether there is a corresponding gene mutation in the tumor cells; and when the replication-deficient adenovirus carrying the suicide gene is used, the anti-tumor effect not only depends on the expression of the suicide gene in the tumor cells, but also depends on the response of the tumor cells to the anti-tumor prodrug Sensitivity of transformed antineoplastic drugs. Therefore, the application of such methods is limited.

Therefore, seeking a more ideal method for in vitro purification of hematopoietic stem cells is still one of the main research directions of scholars in the industry. The present invention is just completed at this demand.

Contents of the invention

Aiming at the state of the art and based on the natural difference of adenovirus receptors on the surface of hematopoietic stem cells and tumor cells, the inventors have carried out a lot of in-depth research work, so that the present invention has finally been completed.

First, the inventors transfected hematopoietic stem cells and tumor cells with a replication-deficient adenovirus (Ad-GFP) carrying the green fluorescent protein gene, and then detected by fluorescence microscopy and flow cytometry, and confirmed that adenovirus has a negative effect on most tumor cells. The transfection rate is high, but it is difficult to transfect hematopoietic stem cells. On the basis of this discovery, the inventor has specifically completed the following invention:

One of the objectives of the present invention is to provide a new method for purifying autologous hematopoietic stem cells in vitro. In the method of the present invention, it includes the steps of contacting hematopoietic stem cells containing tumor cells with genetically engineered proliferative oncolytic viruses and allowing the proliferative oncolytic viruses to enter tumor cells. The tumor cells mentioned therein include blood tumor cells such as multiple myeloma, acute leukemia, chronic leukemia, malignant lymphoma, etc., and non-blood tumor cells such as malignant lymphoma, breast cancer, testicular cancer, ovarian cancer, small Cell lung cancer, nasopharyngeal carcinoma, childhood fibroblastoma, soft tissue sarcoma and other solid tumor cells. The proliferative oncolytic virus includes adenovirus, herpes simplex virus, Newcastle disease virus, vesicular stomatitis virus and reovirus which can selectively proliferate and replicate in tumor cells. The preferred proliferative oncolytic virus of the present invention is a proliferative adenovirus, especially an adenovirus lacking the E1B gene that can be recognized by the adenovirus receptor on the surface of tumor cells. The proliferative adenoviruses include all type 5 proliferative adenoviruses that have lost the E1B gene.

An adenovirus deposited by the applicant in China Center for Type Culture Collection (CCTCC) in 1998 is a type 5 proliferative adenovirus lacking the E1B gene, and its preservation number is CCTCC98003. Hereinafter, CCTCC98003 is sometimes referred to as H101 for short.

In the purification method of the present invention, if tumor cells are used as the target cells for infection, it is preferable to contact the proliferative adenovirus and the tumor cells mixed in the hematopoietic stem cell transplantation in the range of 10 to 10000 MOI (adenovirus/tumor cell), A more preferable range of MOI is 10-5000, and a more preferable range of MOI is 50-1000. When the MOI is lower than 10, due to the low density of adenovirus, individual tumor cells may not be able to fully contact and bind to the adenovirus, so there is a possibility of tumor cells remaining; at this time, some other hematopoietic stem cells can be used Purification methods such as MACS technology or combined with proliferative adenovirus contact again to achieve 100% purification effect. When the MOI is higher than 10,000, it may have some adverse effects on blood reconstitution after hematopoietic stem cell reinfusion; at this time, it may be necessary to administer some drugs or other medical methods that are beneficial to blood reconstitution, thus, it may increase the medical burden on patients .

In the purification method of the present invention, it is preferred that the hematopoietic stem cell graft be contacted with proliferative adenovirus for 30 minutes to 8 hours, preferably 1-4 hours, more preferably 2-4 hours. When the contact time is less than 30 minutes, the combination of tumor cells and adenovirus may be insufficient, resulting in insufficient amount of bound virus, thus affecting the purification effect. When the contact time is longer than 8 hours, not only does it not help to improve the purification effect, but because the hematopoietic stem cells exist for a long time in vitro, the viability of hematopoietic stem cells may decrease.

In the purification method of the present invention, there is no special limitation on the way of contacting the hematopoietic stem cell graft and proliferative adenovirus, and the hematopoietic stem cell graft can be added to the adenovirus preservation solution, or vice versa. Preferably, the adenovirus preservation solution is added to the hematopoietic stem cell transplant.

In the purification method of the present invention, it also includes the step of centrifuging and washing the hematopoietic stem cell graft collected from the patient before contacting the hematopoietic stem cell graft with the proliferative adenovirus. The purpose of centrifugation is to remove serum and platelets, and reduce the influence of serum and platelets on virus-infected tumor cells, and the washing step is to remove residual serum and platelet components. Methods, instruments and reagents for centrifugation, washing and removal of serum and platelets used in the method of the present invention are known to those skilled in the art. Before contacting with proliferative adenovirus, the concentration of hematopoietic stem cells is preferably adjusted to 10 ⁶ -10 ⁹ cells/ml, preferably 10 ⁷ -10 ⁸ cells/ml with cell culture medium.

In the purification method of the present invention, there is no very particular limitation on the mixed volume of hematopoietic stem cells and adenovirus. However, from the perspective of improving the efficiency of mixing and the efficiency of adenovirus binding to tumor cells, it is preferable to mix in a smaller volume, for example, when the MOI is 100-1000, the total volume after mixing is 0.1-100 ml , preferably the total volume after mixing is 0.5-10 ml. When the total volume after mixing is too small, such as less than 0.1 ml or too large, such as exceeding 100 ml, the binding efficiency of adenovirus and tumor cells may be affected.

Another object of the present invention is to provide a kit for purifying autologous hematopoietic stem cells in vitro. The kit for in vitro purification of hematopoietic stem cells of the present invention comprises a reagent containing a proliferative oncolytic virus and an instruction describing the method of using the proliferative oncolytic virus. The proliferative oncolytic virus includes herpes simplex virus, adenovirus, Newcastle disease virus, vesicular stomatitis virus and reovirus which can selectively proliferate and replicate in tumor cells. A preferred proliferative oncolytic virus is a proliferative adenovirus, especially an adenovirus lacking the E1B gene that can be recognized by the adenovirus receptor on the surface of tumor cells. Such proliferative adenoviruses include all type 5 proliferative adenoviruses in which the E1B gene has been deleted, such as CCTCC98003.

The kit of the present invention preferably further comprises reagents for washing the hematopoietic stem cell graft. The washing reagent is generally any appropriate cell culture fluid that can maintain the viability of hematopoietic stem cells, such as RPM1640, or a solution that is isotonic with cells, such as physiological saline.

The kit of the present invention preferably further includes reagents for anticoagulation. The anticoagulant reagents include heparin and sodium citrate.

Kits of the present invention also optionally comprise mixing containers such as test tubes and/or mixing devices such as stir bars or shakers and the like.

Another object of the present invention is to provide a method for treating tumors, comprising the steps of:

1) Obtain hematopoietic stem cell transplants from tumor patients;

2) contacting the hematopoietic stem cell graft obtained in step 1) with a proliferative adenovirus to remove tumor cells in the graft;

3) reinfusing the hematopoietic stem cell graft obtained in step 2) to the tumor patient himself.

In the method for treating tumor patients of the present invention, it is preferable to perform high-dose radiotherapy and chemotherapy on the tumor patients before implementing step 1), so as to kill most of the tumor cells in the patient.

In the method for treating tumor patients of the present invention, it is preferable to centrifuge and wash the hematopoietic stem cell graft to remove serum and platelets therein before step 2). Moreover, before the purified hematopoietic stem cells are returned to the tumor patient, it is preferred to wash them to remove unbound adenovirus. The washing solution used can be cell culture medium such as RPM1640, or physiological saline, etc.

In the above step 2), the tumor cells include blood tumor cells such as multiple myeloma, acute leukemia, chronic leukemia, malignant lymphoma, etc., and also include non-blood tumor cells such as malignant lymphoma, breast cancer, testis Carcinoma, ovarian cancer, small cell lung cancer, nasopharyngeal carcinoma, childhood fibroblastoma, soft tissue sarcoma and other solid tumor cells. The proliferative oncolytic virus includes herpes simplex virus, adenovirus, Newcastle disease virus, vesicular stomatitis virus and reovirus which can selectively proliferate and replicate in tumor cells. A preferred proliferative oncolytic virus is a proliferative adenovirus, especially an adenovirus lacking the E1B gene that can be recognized by the adenovirus receptor on the surface of tumor cells. Such proliferative adenoviruses include all type 5 proliferative adenoviruses in which the E1B gene has been deleted, such as CCTCC98003.

In vitro experiments, the inventors have confirmed that if the proliferative adenovirus CCTCC98003 transfects tumor cells as the target Cells, at least in the range of 100-1000 MOI, can completely eliminate the tumor cells mixed in hematopoietic stem cells, but has no effect on the proliferation ability of hematopoietic stem cells.

In non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mice, through serial pathological sections of tissue and RT-PCR detection of epithelial cytokeratin (CK 19), the purification of tumor cells by proliferative adenovirus CCTCC98003 was completed The evaluation of whether CCTCC98003 affects the hematopoietic reconstitution ability of hematopoietic stem cells was completed through the flow cytometric detection of human blood cells and hematopoietic stem cells in peripheral blood and bone marrow of mice. The results show that CCTCC98003 can completely eliminate the tumor cells mixed in the hematopoietic stem cells under certain dose, mixed culture volume and mixed culture time conditions, but has no effect on the hematopoietic ability of the hematopoietic stem cells.

Unless otherwise stated, the terms and abbreviations used in this specification have the ordinary and conventional meanings used in the art.

Brief description of the drawings

Figure 1 shows the results of fluorescence microscope observation of different types of cells by the replication-defective adenovirus (Ad-GFP) carrying the fluorescent protein gene at 100 MOI. Each type of cells was counted under visible light and fluorescence microscopy in the same field of view, and the ratio of fluorescent cells was calculated as the adenovirus transfection rate. For details, see Fig. 1, Fig. AK, Fig. AY, Fig. BK, Fig. BY, Fig. CK, Fig. CY, Fig. DK, and Fig. DY. The results showed that at the infection titer of Ad-GFP 100MOI, breast cancer cells MCF-7, MDA-MB-231, and multiple myeloma cells SKO-007 could be completely transfected, while peripheral blood hematopoietic stem cells (PBSC) could be transfected even at a MOI of 800 No transfection was seen either.

Figure 2 shows the flow cytometry results of Ad-GFP 100MOI transfection on different types of cells. Flow cytometry found that Ad-GFP was 93.1%, 95.1%, and 86.9% for MCF-7, MDA-MB-231, and SKO-007 at an infection titer of 100 MOI, while the transfection rate of PBSC was as high as The transfection rate at 800MOI was only 1.75%.

Figure 3 shows the 96-hour MTT method to detect the killing effect of CCTCC98003 on different types of cells. The results found that the half tumor inhibitory drug doses (IC ₅₀ ) of CCTCC98003 to MCF-7, MDA-MB-231, and SKO-007 cells in 96 hours were 25, 46.6, and 2.1 MOI virus infection titers, while the control drug Ad- GFP has no obvious killing effect on the above three kinds of cells.

Figure 4 shows the 14-day colony formation method to detect the killing effect of CCTCC98003 on different types of cells. The results found that the half tumor inhibitory drug doses (IC ₅₀ ) of CCTCC98003 on MCF-7, MDA-MB-231, and SKO-007 cells were 2.1, 3.4, and 0.3 MOI virus infection titers, and 100 MOI virus titers were acceptable. Complete tumor growth inhibition was obtained. The control drug Ad-GFP had no obvious killing effect on the above three cells

Figure 5 shows the effect of CCTCC98003 on the formation of CFU-GM in hematopoietic stem cells. It was found that CCTCC98003 had no significant effect on the formation of CFU-GM of hematopoietic stem cells when the MOI was below 800, which was consistent with the results of Ad-GFP.

Figure 6 shows the dose-dependent experimental results of CCTCC98003. Under the conditions of a mixed culture volume of 1ml and a mixed culture time of 2 hours, it can be seen that CCTCC98003 can completely inhibit the growth of MCF-7 cells at a titer of ≥50 MOI.

Figure 7 shows the volume-dependent experimental results of CCTCC98003. When the infection titer of CCTCC98003 is 100MOI and the mixed culture is 2 hours, the growth of MCF-7 cells can be completely inhibited in the range of 0.5-10.0ml. The control was without adding CCTCC98003, and the mixed culture volume was 0.5ml.

Figure 8 shows the time-dependent experiment of CCTCC98003. When the infection titer of CCTCC98003 is 100MOI and the mixed culture volume is 0.5ml, the mixed culture time range of 0.5-8 hours can completely inhibit the growth of MCF-7 cells. The control was without adding CCTCC98003, and the mixed culture time was 2 hours.

Figure 9 shows the simulated purification in vitro experiment of CCTCC98003. 10 ⁵ MCF-7 cells were mixed with 10 ⁷ PBSC cells, and CCTCC98003 was purified at 10, 100, 250, and 500 MOI. It can be seen that CCTCC98003 can completely inhibit the growth of tumor cells when the infection titer of tumor cells is ≥100 MOI. The control drug Ad-GFP had no purification effect on MCF-7.

Figure 10 shows the evaluation of CCTCC98003 simulated purification of hematopoietic reconstitution. Through the CFU-GM formation experiment of hematopoietic stem cells, it was found that CCTCC98003 had no effect on the CFU-GM formation ability of hematopoietic stem cells while purifying MCF-7 cells. Consistent with the results of the control drug Ad-GFP.

Figure 11 is a photo showing the evaluation of the in vitro purification effect of CCTCC98003 (H101) by serial pathological sections of NOD-SCID mice. Continuous pathological sections showed that there were multiple breast tumor nodules in the lungs of the RPM1640 culture solution blank control group and the Ad-GFP control group, but no pulmonary tumor nodules were found in the H101 purification group.

Figure 12 shows the photos of the electrophoresis results of CK19 RT-PCR detection in NOD-SCID mouse lung tissue. It can be seen that there is no CK 19 band at 380bp in the CCTCC98003 purification group, and CK19 is a specific gene marker of human epithelial tumor cells, while the RPM1640 culture medium blank control group and Ad-GFP control group can all see CK 19 amplification bands, Tubulin For internal reference control.

Figure 13 shows the effect of CCTCC98003 purification on the reconstruction of hematopoietic stem cells in NOD-SCID mice. Figure 13A is the result of flow cytometry detection of human-derived blood cells in the peripheral blood of NOD-SCID mice, and Figure 13B is the result of flow cytometry detection of bone marrow human-derived blood cells in NOD-SCID mice, in which CD45 ⁺ cells are human-derived leukocyte markers, CD45 ⁺ CD34 ⁺ cells It is a marker of human hematopoietic stem cells, and CD45 ⁺ CD19 ⁺ cells is a marker of human B lymphocytes. The results showed that CCTCC98003 had no significant effect on hematopoietic reconstitution of human hematopoietic stem cells in NOD-SCID mice.

In order to describe the content of the present invention more clearly, the present invention will be described exemplarily in the form of examples below. These examples do not constitute any limitation to the present invention.

Example 1 Ad-GFP measures the transfection efficiency of hematopoietic stem cells, breast cancer cells, and multiple myeloma cells and counts hematopoietic stem cells PBSC, breast cancer cells MCF-7 and MDA-MB-231, and multiple myeloma cells SKO-007 Cells were inoculated in 35 mm dishes at 10 ⁵ cells/dish, and 500 μl of serum-free 1640 culture medium (purchased from GIBCO, USA) was added to each dish. Add Ad-GFP to the above cell culture dish at a titer of 0, 25MOI, 50MOI, 100MOI, 200MOI, 400MOI, 800MOI, mix and shake at 37°C for 2 hours, add 500μl of 1640 culture solution containing 20% fetal bovine serum, set Into a 37°C CO ₂ incubator, count the Ad-GFP infection rate under a fluorescent microscope after 48 hours, and detect it by flow cytometry. The results show that under the fluorescence microscope, MCF-7, MDA-MB-231, and SKO-007 can obtain 100% transfection at an Ad-GFP 100MOI titer, while for PBSC, even if the Ad-GFP infection titer is as high as 800MOI, there is no Cell transfection was not seen. The results are shown in Figure 1. At the same time, it was found by flow cytometry that the infection rates of MCF-7, MDA-MB-231, and SKO-007 were 93.1%, 95.1%, and 86.9% respectively. The rate is only 1.75%. The results are shown in Figure 2.

Example II Evaluation of CCTCC98003's killing effect on breast cancer cells and multiple myeloma cells

MCF-7, MDA-MB-231, and SKO-007 cells were seeded in 96-well plates with 8×10 ³ cells per well. Add 100 μl of serum-free 1640 culture medium to each well, and transfect Ad-GFP, CCTCC98003 at titers of 0, 25 MOI, 50 MOI, 100 MOI, and 200 MOI respectively, and Ad-GFP was used as a control. Shake and mix in a 37°C incubator for 2 hours, add 100 μl of 1640 culture solution containing 20% fetal bovine serum, put it in a 37°C CO ₂ incubator, and perform MTT assay after 96 hours. MTT detection was performed according to the conventional method, and finally the cell survival rate of different infection titers was calculated. See below for cell viability formula. The dose-cell viability curve was drawn, and the least square method was used for curve fitting, and the IC ₅₀ for inhibiting 50% cell viability was obtained according to the obtained formula.

Cell survival rate (%)=(OD value of drug-added cells-OD value of blank cells)/(OD value of control cells-OD value of blank cells)×100%

After 96 hours of mixed culture, the IC ₅₀ of CCTCC98003 to MCF-7, MDA-MB-231, and SKO-007 were 25, 46.6, and 2.1 MOI respectively. The growth effect of 007 was not obvious. The results are shown in Figure 3: the tumor colony formation method was cultured in vitro for 14 days. It was found that the IC ₅₀ of CCTCC98003 to MCF-7, MDA-MB-231, and SKO-007 were 2.1, 3.4, and 0.3 MOI respectively. 7. The growth effect of MDA-MB-231 was not obvious. The results are shown in Figure 4.

Example III Effect of CCTCC98003 on CFU-GM Formation Ability of Hematopoietic Stem Cells

Freshly isolated PBSC cells were seeded in a 24-well plate with 2000 CD34+ cells per well, added 200 μl 1640, and received 0, 100 MOI, 200 MOI, 400 MOI, 600 MOI, 800 MOI, and 1500 MOI to transfect CCTCC98003 and Ad-GFP. Mix and shake at 37°C for 2 hours, add 400 μl of semi-solid CFU-GM culture medium (purchased from STEM CELL, USA), mix well, and place in a 37°C CO ₂ incubator for cultivation. After 14 days, when visible When the colonies were formed, the culture was terminated, and the number of CFU-GM colonies was counted under a microscope. The colony formation rate was calculated according to the following formula. Colony formation rate = number of colonies / number of seeded cells × 100%

The results showed that when CCTCC98003 was less than 800 MOI, it had no significant effect on the CFU-GM formation ability of hematopoietic stem cells. The results are shown in Figure 5.

Example IVCCTCC98003 Determination of Purification Parameters of Breast Cancer MCF-7 Cells in Vitro

(1) Dose-dependent study. MCF-7 cells in the logarithmic growth phase were taken, counted by trypsinization, and 1000 cells were inoculated in a 60mm dish. Cultivate in a CO ₂ incubator at 37°C. After 24 hours, absorb the serum-containing 1640 culture solution, wash twice with PBS buffer, add 200 μl serum-free 1640 culture solution to each dish, and use 0, 0.001 MOI, 0.01 MOI, 0.1 MOI, respectively. Transfect H101 at 1MOI, 10MOI, 50MOI, 100MOI, 200MOI titers. Shake and mix in an incubator at 37°C for 2 hours, absorb the culture solution, add 4ml of 1640 culture solution containing 10% fetal bovine serum, and place in 37°C CO ₂ After 7 to 14 days in the incubator, when colonies are visible to the naked eye in the culture dish, stop the culture, discard the culture medium, wash twice with PBS buffer, perform 0.2% crystal violet staining for 15 minutes, and slowly rinse the staining solution with water. Dry at room temperature, and count the number of colonies larger than 50 cells under a microscope. It was found that when the dose of CCTCC98003 was 10 MOI, it could significantly inhibit the growth of MCF-7 cells, and when the dose was ≥50 MOI, it could completely inhibit the growth of MCF-7 cells. The results are shown in Figure 6.

(2) Volume dependence study. Tumor colony formation assay was used for evaluation. For CCTCC98003, 100MOI was used as the virus infection titer, and 2 hours was the virus mixed culture time. Under the conditions of different virus-cell mixed culture volumes, the difference in the formation of tumor colonies was observed. The mixed culture volume is selected from 5 different volume units of 500, 1000, 2000, 5000 and 10000 μl. The study of the volume dependence of CCTCC98003 on MCF-7 cells found that at 100 MOI virus infection titer and 2 hours of mixed culture time, the mixed culture volume was in the range of 500-10000 μl, and tumor cells could be completely eliminated. The results are shown in Figure 7.

(3) Time-dependent research. Tumor colony formation assay was used for evaluation. For CCTCC98003, 100 MOI was used as the virus infection titer, and 500 μl was used as the mixed transfection volume of the virus. Under the conditions of different virus-cell mixed culture time conditions, the difference in the formation of tumor colonies was observed. The mixed culture time is selected from 0.5, 1, 2, 4, 8 hours, 5 different time periods. The results show that CCTCC98003 can significantly inhibit the growth of MCF-7 cells in the mixed culture for 0.5 hour, and can completely eliminate the tumor cells mixed in the hematopoietic stem cells when the mixed culture time is ≥ 1 hour. The results are shown in Figure 8.

Example V CCTCC98003 Simulated Purification of Breast Cancer MCF-7 Cells and Hematopoietic Stem Cell Mixture in Vitro

MCF-7 cells were mixed with PBSC cells at a ratio of 1/100 (1×10 ⁴ MCF-7 mixed with 1×10 ⁶ PBSC cells), MCF-7 cells were used as transfection target cells, and 0, 10 MOI, 100MOI, 250MOI, 500MOI titer, transfect mixed culture cells. The volume of virus-cell mixed culture is 500 μl, and the mixed culture time is 2 hours. Finally, the purified mixed cells were evaluated for tumor colony formation and hematopoietic stem cell CFU-GM formation respectively. Tumor cell colonies were evaluated on days 7-14, and CFU-GM were evaluated on days 14-21. The results showed that when MCF-7 was mixed with PBSC at a ratio of 1/100, CCTCC98003 could completely purify tumor cells at an infection titer ≥ 100 MOI, a mixed culture volume of 0.5 ml, and a mixed culture time of 2 hours. The results are shown in Figure 9. And under the above conditions, CCTCC98003 has no effect on the CFU-GM formation ability of hematopoietic stem cells. The results are shown in Figure 10.

Example VI In vivo evaluation of CCTCC98003 purification of breast cancer MCF-7 cells in NOD-SCID mice

A total of 24 NOD-SCID mice aged 6-8 weeks and weighing 20-30 g were selected and randomly divided into three groups of 8 rats, respectively 1640 culture solution blank control group, Ad-GFP control group, and CCTCC98003 purification group. After being mobilized by hematopoietic stem cell stimulating factors, PBSC collected from healthy volunteers were mixed with MCF-7 cells. The mixing conditions were 2×10 ⁵ MCF-7 cells and 2×10 ⁶ PBSC cells, and the total mixing volume was 500 μl. Then, according to the purification results of in vitro experiments, the above mixture was purified using CCTCC98003. CCTCC98003 purification conditions were infection titer 100MOI, mixing volume 500μl, incubation time 2 hours. 500 μl of the purified mixture was injected from the mouse tail vein. Before infusion of the purified mixed cells, the NOD-SCID mice were routinely irradiated with Co ⁶⁰ at a dose of 220cGY to further suppress the immune function of the NOD-SCID mice. Mice were sacrificed on day 45, and venous blood cells and bone marrow cells were collected for human CD45 ⁺ , CD45 ⁺ CD34 ⁺ , CD45 ⁺ CD19 ⁺ flow cytometry to evaluate the ability of human hematopoietic stem cells to reconstruct hematopoiesis in NOD-SCID mice. After the mice were sacrificed, the lung tissue was taken for serial pathological tissue section detection, and CK19RT-PCR detection was performed at the same time to evaluate the purification effect of CCTCC98003 on breast cancer cells. The results showed that in the 1640 culture solution blank control group and the Ad-GFP control group, the continuous pathological sections of the lung tissue of NOD-SCID mice showed multiple breast cancer nodules in the lungs, while no tumor nodules in the lungs were found in the CCTCC98003 purification group. The results are shown in Figure 11. The detection of CK19RT-PCR in the lung tissue of NOD-SCID mice found that the 1640 culture solution blank control group and the Ad-GFP control group could both see the CK19 amplification band with a size of 380bp, while the CCTCC98003 purification group did not see the same size amplification band, showing that CCTCC98003 has a remarkable purification effect. The results are shown in Figure 12.

Through the detection of human CD45 ⁺ cells, CD45 ⁺ CD34 ⁺ , and CD45 ⁺ CD19 ⁺ cells in the venous blood and bone marrow of NOD-SCID mice, it was found that the human There was no significant difference in the levels of CD45 ⁺ cells, CD45 ⁺ CD34 ⁺ , CD45 ⁺ CD19 ⁺ cells, and CCTCC98003 had no significant effect on the reconstitution of human hematopoietic stem cells in NOD-SCID mice. The results are shown in Figures 13A and 13B.

Claims (16)

1. the method for an autologous stem cell purging in vitro comprises:

The hemopoietic stem cell that will contain tumour cell and replication competent adenovirus are that 10～1000 infection multiplicity is mixed according to adenovirus/tumour cell, make described replication competent adenovirus contact and enter the step of tumour cell with tumour cell.

2. the described method of claim 1, wherein said replication competent adenovirus is the 5 type replicative adenovirus that lacked the E1B gene.

3. the described method of claim 1, wherein said tumour cell is selected from malignant lymphatic oncocyte, breast cancer cell, testicular cancer cell, ovarian cancer cell, small cell lung cancer cell, nasopharyngeal carcinoma cell, children's fibroblast oncocyte, soft tissue sarcoma's cell, multiple myeloma cells, acute leukemia and chronic leukemia oncocyte.

4. the described method of claim 1, wherein infection multiplicity is 50～800.

5. the described method of claim 1 wherein contacts the hematopoietic stem cell transplantation thing 30 minutes to 8 hours time with replication competent adenovirus.

6. the described method of claim 5 wherein contacts 1-4 hour with the hematopoietic stem cell transplantation thing with replication competent adenovirus.

7. the described method of claim 6 contacts 2-4 hour with the hematopoietic stem cell transplantation thing with replication competent adenovirus.

8. the described method of claim 1 wherein also is included in the hematopoietic stem cell transplantation thing with before replication competent adenovirus contacts, and the hematopoietic stem cell transplantation thing of gathering in patient's body is carried out step centrifugal and washing.

9. the described method of claim 8 wherein also is included in after the washing step, with physiological saline or cell culture fluid the concentration of hemopoietic stem cell is adjusted into 10 ⁶-10 ⁹The step of individual cells/ml.

10. the described method of claim 9, wherein the concentration of hemopoietic stem cell is 10 ⁷-10 ⁸Individual cells/ml.

11. a test kit that is used for the autologous stem cell purging in vitro is characterized in that comprising:

The reagent that contains replication competent adenovirus,

The specification sheets of replication competent adenovirus using method is described, and optional

Mixing vessel and mixing device.

12. the described test kit of claim 11, wherein said adenovirus are the 5 type replicative adenovirus that lacked the E1B gene.

13. the described test kit of claim 12 also further comprises the reagent that is used to wash hemopoietic stem cell.

14. the described test kit of claim 13, wherein said washing reagent are cell culture fluid.

15. the described test kit of claim 14 wherein also further comprises the reagent that is used for anti-freezing.

16. the described test kit of claim 15, wherein said anti-freezing reagent is heparin or citrate.

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