TWI354024B - Compositions of growth factors for enhancing the w - Google Patents

Description

IX. Description of the Invention: [Technical Field to Which the Invention Is Along] The present invention relates to a human growth factor composition for promoting epidermal cell regeneration and a preparation method thereof. [Prior Art] The skin not only has the functions of regulating body temperature and moisture, but also the first-line defense and line of the human immune system. Once the skin is protected, it is extremely susceptible to foreign pathogen infection. Patients who have experienced burns, ulcers, inflammation, radiation therapy, surgery, and diabetes are prone to infection and cause great harm to the body in the case of skin defects and abnormalities for a long time. Therefore, wound healing and epidermal cell regeneration mechanism is a An important area of medical research. At present, most of the drugs used to treat wounds to promote healing are fungicides, antibiotics, corticosteroids, etc., which have their own shortcomings for wound healing, and the wound healing rate is poor. Therefore, artificial skin or other skin substitutes, cell therapy and the like have been developed. Related studies have indicated that various growth factors play an important role in the wound healing process of the skin. After skin injury, platelets release a variety of growth factors and cytokines, including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and platelet-derived growth. Platelet-derived growth factor (PDGF), human insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), rpm 1354024

Growth factor α (transformation growth factor-α, TGF-c〇, transforming growth factor (jS 'TGF-/3), interleukin-1 'IL-1), connective tissue growth Connective tissue growth factor (CTGF), activin, tumour necrosis factor (TNF-c〇, etc. These growth factors can attract neutrophil and macrophage. Gathering toward the wound to remove the injured tissue, cells and phagocytosis of the infection, it can also activate nearby fibroblasts and keratinocytes and induce peripheral blood vessels to undergo angiogenesis.

On the other hand, fibroblasts at the wound site are transformed into myofibroblasts under the influence of certain growth factors (especially PDGF and TGF-cold). These myofibroblasts express a large amount of alpha actin (a-actin) to contract the wound and synthesize collagen fibers. The arrangement of these collagen fibers deposited on the wound is affected by the contraction of the myofibroblasts and is different from the collagen fibers of the original normal tissue, thereby forming scar tissue. The collagen in the scar tissue is rearranged from the fourth week after the injury to about six months, and the shape and function of the scar tissue are close to the original normal tissue. Recently, studies have indicated that TGF-/33 regulates wound healing not only for macrophages and fibroblasts, but also stimulates the transcription of mRNA of type I and type III collagen to promote wound healing. The accumulation of collagen in order to improve the healing rate of the wound. Therefore, TGF-/33 has been shown to produce innocuous epithelial regeneration and accelerate healing of skin wounds caused by radiation exposure. Studies have shown that a large amount of TGF-|33 can be secreted during the embryonic period of mammals. However, when the epidermis of adult mammals is injured, the amount of (S) 6 1354024 TGF-/53 secreted is very small. After the wound, it is easy to have scars caused by contraction of muscle fiber cells. Growth factors play an important role in the regeneration and repair mechanisms of wounds, including EGF, bFGF, IGF-1, PDGF, and VEGF.

They have been widely used in medicine and beauty care products#. However, at present, genetic engineering is used to prepare a large amount of the above growth factors, and genes expressing the above human growth factors are individually inserted into a vector to multiply and further purify the human growth factor. Although this method can reduce the cost, the disadvantage is that the purified protein has different purity and may contain endotoxin or other undesirable proteins, which may affect the therapeutic effect or complicated allergic reaction; in addition, one plastid is inserted at the same time or more. The growth factor genes are all active and difficult to perform. It is time-consuming and labor-intensive to operate multiple plastids at the same time. Moreover, when prokaryotic or fungi are used to express human growth factor proteins, most of them The activity of the produced protein is reduced. On the other hand, stem cell therapy utilizes human adult stem cells, such as stem cells located in the bone marrow, epidermis, and intestines, to treat various diseases. The human mesenchymal stem cell 'hMSC' isolated from human bone is an adult stem cell with various differentiation potentials, which can differentiate into bone, cartilage, muscle, skin and other tissues in different culture environments. Easy to separate 'culture and in vitro proliferation speed characteristics, has been confirmed by many studies to strengthen the skin wound healing mechanism. However, transplantation into the human body by stem cell injection or surgery requires long-term clinical trials to assess whether allogeneic stem cell transplantation produces immune rejection. Furthermore, the medical resources and money spent on stem cell treatment are also in general therapy. Therefore, there is a need to propose a method that can be combined with genetic engineering and stem 1354024 to require a method that can be both long-term and permeable to promote the advantages of epidermal cell therapy and improve its disadvantages. It is also possible to provide three or more types of growth by human cells, generally for external or internal administration. SUMMARY OF THE INVENTION In view of the above problems, the present invention proposes a method for culturing human bone leaf stem cells in vitro and/or preparing human growth factors. First, the primary culture of human bone marrow mesenchymal stem cells was carried out in the primary #A culture solution. Next, the suspension cells in the primary culture medium are selected and the adherent cells are selected and subcultured by subculture. Thereafter, the adherent cells are selected and cultured in the final culture medium, which is a serum-free culture solution containing human growth hormone. The last culture allows the adherent cells to secrete a variety of growth factors into the final culture to form a human growth factor solution. That is, the above human growth factor solution contains at least a combination of a plurality of human growth factors secreted by human bone marrow stem cells, i.e., the human growth factor composition referred to in the present invention. The human growth factor composition described above comprises at least three growth factors selected from the group consisting of EGF, bFGF, IGIM, PDGF, VEGF, TGF, stem cell growth factor (Claw _ faCt〇r 'SCF), interferon (interferon, IFN-γ), chemokine (RANTES), platelet growth factor (thr〇mb〇p〇ietin, Tp〇), tissue inhibitor of metalloproteinase (tissue), metalloproteinase (TIMP), leptin ) 'Interleukin-6 'IL-6', and interleukin_8, IL-8. According to an embodiment of the present invention, a nutrient solution can be added to the above-mentioned last culture solution selectively 1354024. The nutrient solution may comprise at least one amino acid and/or at least one vitamin. According to an embodiment of the present invention, the above human growth factor solution can be taken out and dialyzed to purify the above human growth factor solution.

According to still another aspect of the present invention, there is provided a composition for promoting regeneration of epidermal cells, the composition comprising at least the human growth factor composition, the nutrient component, the emulsifier solution, and the oil phase liquid of the embodiment of the present invention. The above nutrient component comprises a portion of the nutrient solution used in the preparation of the human growth factor component in accordance with an embodiment of the present invention. The emulsifier solution and the oil phase liquid can embed the human growth factor composition and nutrient components in a water-in-oil in water (w/o/w) manner into a micro-lipid body embedding material. . According to an embodiment of the present invention, a water-soluble colloid can be further added to the above composition for promoting epidermal cell regeneration to increase the stability of the above-mentioned liposome-embedded material.

By using the method for in vitro culture of human bone marrow mesenchymal stem cells in the present invention, a human growth factor composition can be easily obtained, and the type, activity and safety of the human growth factors contained therein are higher than those produced by genetic engineering. Further, the growth factor composition obtained by the method according to the embodiment of the present invention comprises at least a plurality of growth factors associated with epidermal cell regeneration, which can effectively enhance the ability of epidermal cells to regenerate. In addition, the nutrient solution according to the embodiment of the present invention not only provides additional nutrients to the human bone marrow mesenchymal stem cells during the preparation of the human growth factor component, but also contributes to enhancing the combination of the present invention for promoting epidermal cell regeneration. The human growth factor contained in the composition contributes to the ability of epidermal cell regeneration 9 1354024. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In order to make the above-mentioned objects, features, and preferred embodiments of the present invention, the detailed description will be

A method for cultivating a human stem cell and/or preparing a human biosynthesis outside Zhao Zhao. The present invention provides a method for culturing a human bone mesenchymal stem cell in vitro, the method comprising the following steps: a) primary culture of human bone marrow mesenchymal stem cells in primary culture medium; (b) removal of suspension cells in primary culture medium and selection of adherent cells, and subculture with subculture medium;

(c) Select the adherent cells in the subculture medium and use the final culture solution for the final culture. The above-mentioned last culture medium is a serum-free medium and contains a human growth factor to enable the adhesion cells to secrete various growth factors into the final culture solution to form a human growth factor solution in a specific embodiment of the present invention. Healthy human bone marrow mesenchymal stem cells (purchased from The National Disease Research Interchange, NDRI, USA) in dMEM medium containing 10% fetal bovine serum (Dulbecco, s modified eagle medium, available from Gibico, Cat· Νο·12100-061 As a primary culture solution, the primary culture was carried out for 3 days. Then, the suspension cells in the primary culture 1354024 solution were removed and the adherent cells were selected, and the DMEM culture solution containing 1% fetal calf blood/month was used as the subculture medium, and subculture was carried out for 3 days. • After subculture, the last culture medium was cultured for 3 days. The above-mentioned terminal culture medium was a serum-free medium containing about 5 to 25 ng/ml of human tamsin growth factor. In the final culture step, the adherent cells in the final culture solution can secrete a plurality of human growth factors into the final culture solution to form a human growth factor solution. These human growth factors contain at least three of the following growth factors: EGF, bFGF, IGF-1, PDGF, VEGF, TGF-/33, SCF, IFN-γ, RANTES, TP〇, TIMP, leptin' IL-6, And IL-8. The combination of these human growth factors is referred to as "human growth factor composition" in the examples of the present invention. Furthermore, according to an embodiment of the present invention, the concentration of the growth factor contained in the human growth factor composition is detectable, and the detection mode and detection limit will be described in detail below. According to a specific embodiment of the present invention, the subculture step can be repeated up to 20 times as needed. According to a specific embodiment of the present invention, the cultured human bone marrow mesenchymal stem cells can be cryopreserved after the subculture step φ, and the cryopreserved human bone marrow mesenchymal stem cells can be subcultured after being thawed. However, the number of subcultures of human bone marrow mesenchymal stem cells after thawing can be repeated up to 3 times. In a specific embodiment of the invention, a nutrient solution may be added to the final culture solution, the nutrient solution comprising at least one amino acid and/or at least one vitamin. The above amino acid may be, for example, alanine, arginine, aspartic acid, aspartic acid, cysteine, cystine, glutamic acid, glycine, histidine, isalamine. Acid, leucine, lysine citrate, amphetamine 11 jr/p valine, serine, sulphate, tryptophan, tyrosine or valine, selected parent amine The concentration of the base acid is about 5 to 100 mg/L. The above vitamins may be, for example, vitamin A, vitamin B, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, vitamin C, vitamin D2, vitamin E, vitamin H, vitamin K3, gasified choline folic acid, or muscle. Alcohol, and each selected vitamin has a concentration of about 0.1 to 10 mg/L. In one embodiment of the invention, a human growth factor solution prepared in accordance with an embodiment of the invention is dialyzed to further purify the human growth factor. According to an embodiment of the present invention, a dialysis membrane (purchased from Pierce, SnakeSkinTM Pleated Dialysis Tubing, 35 MWCO) of a 3·5 kd is used for dialysis of the above human growth factor solution, and the dialysis can be continued. 3 to 5 days. The substance having a molecular weight of less than 3,5 Dalt〇n (D) in the human growth factor solution can be substantially removed by the dialysis step, thereby achieving the purpose of purifying the human growth factor solution. Since the molecular weight of the human growth factor contained in the human growth factor composition to be prepared according to the embodiment of the present invention is much larger than 35 KD, for example, the molecular weight of egf is about 6 KD and the molecular weight of TGF-positive is as high as 25 KD, so Most of the human growth factors produced by the methods of the embodiments of the present invention remain in the dialysis membrane. Finally, the solution remaining in the dialysis membrane is collected, which contains at least two human growth factors. Such a human growth factor composition prepared by the method of the present invention is characterized by comprising at least three detectable growth factors (detection methods will be described in detail below), and the growth factors may be EGF, bFGF, IGF- 1. PDGF, VEGF, TGF, SCF, IFN-γ, RANTES, Τρο, TIMP, leptin, IL-6, or IL_8. <S) 12 1354024 Example (II) Qualitative analysis of human growth factor composition

The growth factor composition in the dialysate was measured by RayBioTM Human Cytokine Antibody Arrays, and the results were as shown in the microarray of human interleukin antibody in Figure 1. It can be found from Fig. 1 that the human growth factor composition and its limit of detection (LoD) contained in the human growth factor composition prepared according to the embodiment of the present invention are as follows: EGF (detection limit 1 pg/ml) , bFGF (4 极限 limit 10000 pg/ml), IGF-1 (detection limit 10 pg/ml), PDGF (detection limit 1000 pg/ml), VEGF (detection limit 100 pg/ml), TGF- /33 (detection limit 100 pg/ml), SCF (detection limit 10 pg/ml), IFN-γ (detection limit 100 pg/ml), RANTES (detection limit 2000 pg/ml), Τρο (detection Test limit 100 pg/ml), TIMP (detection limit 1 pg/ml), steroid voxel (detection limit 100 pg/ml), IL-6 (detection limit 1 pg/ml), and IL-8 ( The limit of the debt test is 1 pg/ml).

In the present specification, the term "detection limit" means the lowest detection concentration of the detection method used in the qualitative analysis of human growth factors by the method of the embodiment of the present invention. In other words, the concentration of a particular human growth factor contained in the sample to be tested must be at least equal to or greater than the detection limit used by the detection method before it can be detected. In this case, the specific human can also be Growth factors are called "detectable" human growth factors.

In addition, the SCF RayBioTM ELISA Kit kit and the VEGF RayBioTM ELISA Kit kit were used to quantify both SCF and VEGF growth factors, and the results are shown in Figures 2A and 2B. It can be found from the second and second graphs that the concentration of SCF in the human growth factor composition prepared in the examples of the present invention can be determined by using the SCF and VEGF (S) 13 1354024 standard attached to each set as a regression line. 1380 pg/mi, and the concentration of VEGF is about 25130 pg/ml. Compared to the stem cell culture method used in the prior art, the culture solution contains no detectable TGF-/?3 (detection limit 1 〇〇pg/ml) and sCF (detection limit 10 pg/ml). That is, using the prior art stem cell culture method, the content of TGF-3 in the culture solution is less than 丨〇〇pg/mi, and the sCF content is less than! 〇pg/m Bu Further 'Using the prior art stem cell culture method, the concentration of VEGF in the culture solution is only about 200-500 pg/m. However, the human growth factor solution of the embodiment of the present invention contains detectable TGF_. The content of TGF-/33, which is 1〇〇pg/ml or more, and the concentration of SCF (1380 pg/ml) and VEGF (2513〇pg/ml) are much higher than those of the prior art. EXAMPLES (III) Composition for Promoting Epidermal Cell Regeneration In another embodiment of the present invention, a composition for promoting epidermal cell regeneration comprising at least a human growth factor composition according to an embodiment of the present invention is provided Nutrient component, an emulsifier solution, and an oil phase liquid. The above nutrient component comprises a part of a nutrient solution used in the preparation of a human growth factor composition according to an embodiment of the present invention. In a preferred embodiment, the nutrient component comprises at least one More or more amino acids and/or vitamins. The above emulsifier solution and oil phase liquid can embed human growth factor composition and nutrient components in a water-in-oil/water-in-water (w/0/w) manner into a liposome-embedded material. 1354024 In the embodiment of the present invention, the emulsifier solution comprises about 0.5-1%

Tween 80, and about ι_ι% sorbitan sesquioleate (Arlacel 83). In an embodiment of the invention, the emulsifier solution may further comprise from about 80% to about 90% pure water, from about 1-1% hyaluronic acid, and/or from about 1-10% glucose.

In an embodiment of the invention, the oil phase liquid comprises from about 1-10% egg yolk, from about 1-10% cholesterol, and from about 80% to 90% phospholipid. In the examples of the present invention, the above phospholipids can be replaced with other oil and fat components, for example, the above oil phase liquid may comprise sesame oil and/or mineral oil. According to an embodiment of the present invention, a water-soluble colloid can be added to the above composition for promoting epidermal cell regeneration to increase the stability of the liposome embedding. The water-soluble colloid comprises about 1-10% Tween 20, about 1-10% propylene glycol (PG), about 1-10% PEG-600, and about 1-5% colloid, wherein the colloid The substance may be Sanxian gum, clear gum, or a combination thereof. In one embodiment of the invention, the water soluble colloid may further comprise from about 1% to about 10% hyaluronic acid.

According to one embodiment of the invention, about 2-5 grams of Tween 80, about 2-5 grams of Arlacel 83, about 3-5 grams of hyaluronic acid, and about 2 are added to about 1 inch of pure water at about 4 °C. 5 grams of glucose was dissolved to prepare an emulsifier solution having a total volume of about 20 ml. On the other hand, about 5 to 10 grams of lecithin, and about 2 to 5 grams of cholesterol are added to about 20 to 30 ml of castor oil and about 10 to 20 ml of mineral oil to prepare an oil phase liquid having a total volume of about 40 ml. Further, in about 60-80 ml of pure water, about 10-20 grams of Tween 20, about 10-20 grams of PG, about 2-5 grams of PEG-600, about 5-10 grams of Sanxian gum, and about 10- 20 g of hyaluronic acid was prepared to prepare a water-soluble colloid having a total volume of about 120 ml 15 1354024. About 20 ml of the emulsifier solution and about an equal volume (i.e., about 2 〇 mi) of the 3.5 KD dialyzed dialyzed human growth factor solution were mixed at about 4. Next, about an oil phase liquid, such as an oil phase liquid, is slowly added to a high pressure homogenizer of 8, ·1 〇, _ Γ ρηη at about 4. (: Stir well, stir for about 1〇2〇 minutes. To form micro-lipid nanoparticles (w/0/w)<> Finally, add about 12〇〇1丨 water-soluble colloid to 2,500 The 3,000 rpm homogenizer is stirred uniformly at about 4 Torr, and the stirring is continued for about 20 minutes. The resulting chylomorphous composition is the composition for promoting epidermal cell regeneration in the examples of the present invention. In addition, the purpose of adding a water-soluble colloid is to increase the stability of the micro-lipid nanoparticle, and T keeps the water-soluble protein of the liposome nanoparticle (4) at room temperature for 2 to 3 years. Mouse Skin Wound Healing/Epidermal Cell Regeneration Test In a specific embodiment of the present invention, the effect of the composition for promoting epidermal cell regeneration on wound healing rate in the mouse skin wound healing test is measured. A total of two experimental groups (microlipid A, and liposome B) and one control group (lipid c) were used in this experiment. Each group used four Balb C rats, eight weeks old, to Each mouse was sterilized with a scalpel to cut the back of the mouse after shaving Kneeling.5 χ 〇·5 cm2 wound, then observe the small rolled skin wound with a microscope, and take and record the wound image of the third day, and calculate the wound area on the third day of the soft body; the test of mouse skin wound healing test The design is as follows: Lipid A: The human raw 1354024 long factor solution described in the examples of the present invention is embedded in a liposome (ie, the composition of Example (3) for promoting epidermal cell regeneration). : The nutrient solution of the embodiment of the present invention is embedded in a liposome C: a physiological saline solution is embedded in a liposome.

Approximately 100 μL of liposome A, B, or C was evenly applied to the wound of the test mouse one hour after the wound was caused. Thereafter, about 100 μM of the liposome A, B, or C was applied in the same manner every 24 hours, and applied a total of three times. Twenty-four hours after the third application, that is, the third 曰 after the wound was produced, the skin wound of the mouse was observed under a microscope, and the image of the wound on the third day was taken and recorded, and the wound area on the third day was calculated by software. The microscope used in this embodiment is connected to a charge coupled device (CCD) to capture the image of the mouse skin wound, and after capturing the image, the image is transmitted to a computer for image processing and injury alpha area calculation, Statistics; the software used for image processing and wound area calculation is Northern Eclipse image system; while data statistics are software

The SigmaStat program (2002) performed the analysis and performed the leaf calculation using the Duncan's New Multiple Range Test (DMRT) of the One-way Analysis of Variance (ANOVA). The effect of mouse skin wounds is shown in Figure 3. Figure 3A shows the wound image of the mouse Dijon; Figure 3B shows the wound image of the mouse treated with the liposome A on the 3rd day; and 3C shows the 3rd day of the mouse treated with the liposome B. The wound image; and the 3D image is the wound image of the third day of mice treated with liposome C. Comparing Figures 3A-D, it was found that the composition (microlipid A) treated by the embodiment of the present invention to promote epidermal cell regeneration = the skin wound area of the mouse after day (as shown in Fig. 3B), Less than the other group (S) 17 1354024 The mouse skin wound area (as shown in Figures 3C and 3D). The skin wound area on day 0 and day 3 of each group of mice was calculated by image processing software to convert the wound healing rate' as follows: Wound healing rate (%) = (day 0 skin wound area, day 3 Skin wound area) / Dijon skin wound area x 100% The average wound healing rate of each group of mice was taken. The results are shown in Fig. 4, in which the wound healing rate of the mice in the liposome group A was about 70%. The wound healing rate of the liposome B mice was about 61%, and the wound healing rate of the mice in the liposome group C was about 52 〆. The statistical analysis results of the wound healing rate are shown in Table 1. From Table 1, it can be found that compared with the control group, the liposome c, the test group, the liposome A and the liposome B, the wound healing rate of the mice was significantly different (p < 〇 〇 5). --;_:- ·" 'Heart w like mouth ST ST ST ST ST / / Γ wound healing rate (%) I II III IV liposome A 69 68 73 71 microlipid B 61 60 62 60 microlipid C 52 51 49 54 60.75 0.96 51.50 2.10

EXAMPLES (V) Human skin test, Example - Preparation of liposome A: Embedding human growth factor solution according to the embodiment of the present invention with a liposome (to promote epidermal cell regeneration 18 1354024)

The composition was subjected to human skin test. The data of the test subjects are shown in Table 2. The test sites of each test subject were photographed before the test, as shown in Figures 5A' 5C, 5E, and 5G. Table 2 Human skin test test data Tester Gender age test site Test before tester Tester 1 Male 44 Eye wrinkles 5A Figure 5B Figure 2 Male 29 Forehead pimples 5C Figure 5D Tester 3 Female 20 Face Pimples 5E Figure 5F Figure Tester 4 Female 25 Face Youth Epidemic 5G Figure 5H

The microlipid A prepared in the above Example 3 was partially applied to the test site of each tester, '2 times per smear', 2 ml was applied once, and after 3 s continuously, the test site of each test subject was photographed as shown in the first section. 5B, 5D, 5F, and 5H are shown. As can be seen from Figures 5A and 5B, the use of the composition for promoting wound healing provided by the present invention can reduce facial wrinkles. In addition, as can be seen from the 5D, 5F, and 5H diagrams, the composition for promoting wound healing provided by the present invention can also cause scarring of the skin of the face (e.g., desalination of juvenile cancer. It is known from the preferred embodiment of the present invention described above) The application of the present invention has the following advantages: First, 'the method for culturing human bone marrow mesenchymal stem cells in vitro by the embodiment of the present invention' enables human bone marrow mesenchymal stem cells to produce human growth factor 19 1354024, and the above human growth factor composition includes at least plural Growth factors associated with wound healing/epidermal cell regeneration. In addition, since these growth factors are expressed by human bone marrow mesenchymal stem cells, the components are the same as the human body's own growth factors and contain no protoxin. The activity and species of these growth factors are also superior to those of human adult cells and/or genetically modified xenogenic cells.

Furthermore, according to the culture method of the embodiment of the present invention, a nutrient solution containing one or more amino acids and/or vitamins may be added to the final culture solution to provide additional nutrients to the human bone marrow during the culture process. Mesenchymal stem cells. Further, when the composition for promoting epidermal cell regeneration is prepared according to the present invention, it is not necessary to remove the nutrient solution additionally added in the last culture, and thus the nutrient solution contained in the composition for promoting epidermal cell regeneration can be additionally provided. The epidermis, dermal tissue regeneration, and nutrients required for collagen fiber reforming, and the human growth factor composition of the present invention produces a multiplying effect to further enhance wound healing and/or collagen fiber reformation of epidermal and dermal tissues. ability. Finally, the above composition for promoting epidermal cell regeneration can be formed not only by the liposome entrapment method to form a growth factor liposome which can be used internally or externally, but also can be added to an artificial skin or a skin substitute. To promote epidermal cell regeneration. While the present invention has been described above by way of a preferred embodiment, it is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Figure 2A illustrates the concentration of stem cell growth factor (SCF) in the growth factor composition of the present invention; Figure 2B illustrates the concentration of vascular endothelial growth factor (VEGF) in the growth factor composition of the present invention;

3rd AD is a photomicrograph illustrating the healing of a mouse skin wound in accordance with an embodiment of the present invention; and FIG. 4 is a bar graph illustrating the treatment with various liposome components in accordance with an embodiment of the present invention. The effect of mouse skin wounds on the rate of skin wound healing. Fig. 5A-Η is a photomicrograph illustrating the case of human skin wound healing and lightening of fine lines in accordance with an embodiment of the present invention.

[Main component symbol description] 21

Claims (1)

1354024 Dec. I. Scope of application: 1 · A method for culturing human bone marrow mesenchymal stem cells in vitro to prepare human growth factors, the method comprising at least: performing primary culture in a DMEM containing 10% fetal bovine serum The human bone marrow mesenchymal stem cells are cultured in the primary culture medium of the culture solution; the adherent cells in the primary culture medium are taken out, and subcultured in a subculture medium containing D〇 culture medium containing 10〇/〇 fetal bovine serum; The adherent cells in the subculture medium are cultured in a final culture medium, and the adhesive cells in the last culture medium secrete various growth factors into the final culture solution to form a human growth factor solution. The last culture medium is a serum-free medium and contains at least a human insulin growth factor at a content of about 5 to 25 ng/ml; and the "sino growth factor solution is taken out and dialyzed using a 35 〇〇 kD dialysis membrane 3 to 5 The method of purifying the human growth factor solution. The method of claim 1, wherein the method of the first generation culture, wherein the subculture is 2. Please follow the steps of the patent scope to carry out the following steps: • If the application of the patent scope is carried out, the steps are carried out. 3. If the application of the patent scope is the step of the subculture, the method described in the first paragraph Further, the method includes repeating the number of repetitions up to 20 times. 22 1354024 Modified on May 26, 100. The method of claim 5, wherein the last cultivation step is carried out for 3 days. The method of claim 1, wherein the last culture solution further comprises a nutrient solution, wherein the nutrient solution comprises at least: 'at least one amino acid selected from the group consisting of alanine, arginine, and Tianmen Aspartic acid, aspartic acid, hemi-deaminic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, amphetamine ^ &amp;, a group consisting of proline, serine, threonine, tryptophan, tyrosine, and valine; and/or to &gt;, vitamins selected from vitamin A, Vitamin b 1, vitamin B2, vitamin B3, vitamin ^, vitamin lang, a group of biotin Bn T, vitamin D2, vitamins (4), vitamin H, vitamin κ3, gas choline, folic acid, and inositol. 7. The method of claim 6 , wherein the at least acid content is about 5 to (10) mg / L, and the at least - vitamin content is about 1. 1~10 mg / L. 8. - Human growth factor composition, its utilization Prepared by the method according to any one of claims 1 to 7, which comprises at least three measurable growth factors selected from the group consisting of epidermal growth factor, basic fibroblast growth factor, human insulin. A group consisting of growth factors, platelet-derived growth factors, gold tube endothelial growth factor, transforming growth factor, and stem cell growth factor. 23 1354024 Modified on May 26, pp. 9 - A composition for promoting regeneration of epidermal cells, comprising: - a human growth factor composition as described in claim 8; - a nutritional component comprising at least a monobasic acid and/or at least one vitamin, wherein the amino acid is selected from the group consisting of alanine, arginine, aspartic acid, aspartic acid, cysteine, cystine, glutamic acid, Glycine, group 'amino acid, isoleucine, leucine, lysine, methionine, phenylalanine, valine, serine, threonine, tryptophan, tyrosine, And a group consisting of proline, which is selected from the group consisting of vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, vitamin C, vitamin D2, vitamin E, vitamin H a group consisting of vitamin K3, gas choline, folic acid, and inositol; an emulsifier solution; and an oil phase liquid, wherein the emulsifier solution and the oil phase liquid constitute the human growth factor and the nutrient Ingredients are packaged in a water-in-oil-in-water package To a liposome composition entrapping D # 1〇 as the scope of the patent application to facilitate reepithelialization composition of item 9, wherein the emulsifier solution comprises Tween 80, and sorbitan oleate grease. 11. The composition for promoting epidermal cell growth as described in claim 9 wherein the emulsifier solution further comprises pure water, hyaluronic acid, and glucose. 12. A composition for promoting the regeneration of epidermal cells as disclosed in claim 9 of the ninth aspect of the invention, wherein the oil phase liquid comprises lecithin, cholesterol and squamous lipids. 13. The composition for promoting epidermal cell regeneration according to claim 9, wherein the oil phase liquid further comprises castor oil, or mineral oil, 14', as described in claim 9 The composition for promoting regeneration of epidermal cells further comprises a water-soluble colloid, wherein the water-soluble colloid comprises Tween 20, propylene glycol, PEG_6 oxime, and a colloidal substance, wherein the colloidal substance is Sanxian gum, transparent gum, or a combination thereof. Things. 15. The composition for promoting epidermal cell regeneration as described in claim 14, wherein the water-soluble colloid further comprises hyaluronic acid. 25 1354024 c % V. ('ontrol IFN rl(;F1,Φ IFN 7 IGF 1 H6 PI(,F FK;t mvn:s S(下,· RAMKS S(T IGF M * IUf US ΓΙΜΡ 2 UA' ΕΝΛ -Ί hFGF • t OT ENA fly hFGF 4 • IJ: PTLS. PIX; FBB * • ίΙΡΠΝ PIX; F fiii 警 · \T.GF Thromhopoiffin ΜΧΛ IGF ΓΙ.ΜΡ 1 Ihromhopoietin % Omuol (oriiro! t Figure 1 1354024 Stem cell growth factor 10 - y=0.4373x+0.0039 OSKIO 0· 〇 v.yjyji 0.1 Pg/ml 2A §αο •° vascular endothelial growth factor 0· 00 ο Figure B 2 (S ) 1354024 L_ % Figure 3A Figure 3B Figure 3C Figure 3D 1354024 ο ο ο ο ο ο ο 8 7 6 5 4 3 2 (%) 铢 &lt;o#Dffve hide ac ο group liposome C microlipid B liposome A # Figure 4 1354024 fj as (4) correction replacement 9 Figure 5A Figure 5B Figure 5C Figure 5D Figure 5E Figure 5F Figure 5G Figure 5H