阅读说明：本技术 一种促进子宫内膜修复的干细胞复合蛋白制剂的制备方法 (Preparation method of stem cell composite protein preparation for promoting endometrial repair ) 是由 胡储涵 于 2020-05-26 设计创作，主要内容包括：本发明涉及一种促进子宫内膜修复的干细胞复合蛋白制剂的制备方法。本发明的制备方法步骤如下：1)人脐带间充质干细胞的原代提取和培养；2)人脐带间充质干细胞特异性诱导培养；3)干细胞特异性蛋白复合物上清的制备和浓缩；4)干细胞浓缩因子的冻干；5)壳聚糖温敏凝胶的制备。本发明具有取材便利、细胞得率高、生产过程易控制及生产成本低的优点。(The invention relates to a preparation method of a stem cell complex protein preparation for promoting endometrial repair. The preparation method comprises the following steps: 1) primary extraction and culture of human umbilical cord mesenchymal stem cells; 2) specific induction culture of human umbilical cord mesenchymal stem cells; 3) preparing and concentrating a supernatant of the stem cell specific protein complex; 4) freeze-drying the stem cell concentration factor; 5) preparing the chitosan temperature-sensitive gel. The invention has the advantages of convenient material taking, high cell yield, easy control of the production process and low production cost.)

1. A preparation method of a stem cell complex protein preparation for promoting endometrial repair is characterized by comprising the following steps: the method comprises the following steps:

1) primary extraction and culture of human umbilical cord mesenchymal stem cells:

1.1) extraction of human umbilical cord mesenchymal stem cells: the umbilical cord tissue is obtained from healthy newborn at term, and after tissue sterilization, Wharton's jelly is peeled off and cut into 0.5cm³Centrifugally cleaning tissue blocks with the sizes, adding the tissue blocks into a culture medium, performing conventional culture in an incubator with the temperature of 37 ℃ and the concentration of 5% CO2, replacing the culture medium every 3 days, removing umbilical cord tissues after about 12-14 days, and performing adherent cell culture;

1.2) when the cell fusion degree reaches a certain degree, digesting the cells by using 0.25% pancreatin-EDTA and carrying out passage;

2) specific induction culture of human umbilical cord mesenchymal stem cells:

taking suitable substitute secondary cells, when the cells are cultured to a certain fusion degree, discarding the original culture medium, washing for 3 times by using sterile normal saline, replacing the cells with a serum-free culture medium containing a ferric ion chelating agent Deferoxamine (DFO) for pretreatment culture, and collecting culture supernatant after culture;

3) preparation and concentration of the supernatant of the stem cell-specific protein complex:

3.1) centrifuging the collected culture supernatant to remove cell debris;

3.2) carrying out primary ultrafiltration by using a filter membrane, and then replacing the filter membrane to concentrate the filtrate to obtain primary concentrated solution.

3.3) washing the first concentrated solution twice by using sterile normal saline and performing ultrafiltration again by using a filter membrane, wherein the aim is to completely remove the iron ion chelating agent deferoxamine from the concentrated solution, and the finally obtained concentrated solution is the stem cell concentrated factor and is stored at the temperature of minus 80 ℃;

4) freeze-drying of stem cell concentration factor:

measuring the protein concentration of the obtained stem cell concentrated factor, and adjusting the proportion (mass ratio) of each excipient according to the measurement result; adjusting the concentration of the stem cell concentration factor with sterile water for injection, filtering with a 0.22um filter membrane, and then subpackaging and freeze-drying at a rate of 2ml per branch. Freeze-drying to obtain stem cell concentration factor freeze-dried powder with the concentration of 0.2 mg/branch;

5) preparing the chitosan temperature-sensitive gel:

5.1) preparing chitosan: adding medical chitosan powder into acetic acid, stirring with a magnetic stirrer, and standing at 4 deg.C for storage;

5.2) sodium Beta-glycerophosphate (Beta-Glycerol phosphate, Beta-GP) configuration: dissolving beta-GP in sterile water for injection, shaking and stirring, filtering by a 0.22 mu m filter, preparing a solution, and standing and storing at 4 ℃;

5.3) preparing the chitosan temperature-sensitive gel: taking the prepared chitosan and beta-GP out of the environment at 4 ℃, gradually dripping the prepared chitosan and beta-GP solution on ice into the chitosan solution, continuously stirring to obtain the chitosan temperature-sensitive gel, subpackaging the chitosan temperature-sensitive gel into penicillin bottles by 3 ml/bottle, and storing at 4 ℃.

2. The method for preparing a stem cell complex protein preparation for promoting endometrial repair according to claim 1, wherein the stem cell complex protein preparation comprises: in the step 1.1), the centrifugation condition is room temperature, the centrifugation is carried out for 4-7min under the rotation speed condition of 200g-450g, and the added culture medium after the centrifugation and cleaning is DF-12 culture medium containing 10% volume fraction fetal calf serum.

3. The method for preparing a stem cell complex protein preparation for promoting endometrial repair according to claim 2, wherein the stem cell complex protein preparation comprises: in the step 1.2), cell digestion is carried out when the cell fusion degree reaches 70-80%.

4. The method for preparing a stem cell complex protein preparation for promoting endometrial repair according to claim 3, wherein the stem cell complex protein preparation comprises: in the step 2), the cells suitable for generation are from generation P3 to generation P6; the concentration of iron ion chelating agent Deferoxamine (DFO) was 100. mu.M-200, and the serum-Free medium was MSC Xeno-Free SFM (Corning Cat. No. 88-600-CV), and culture supernatant was collected after 48 hours of culture.

5. The method for preparing a stem cell complex protein preparation for promoting endometrial repair according to claim 4, wherein the stem cell complex protein preparation comprises the following steps: in the step 3.1), the centrifugation conditions of the collected supernatant are 300g-500g at 4 ℃ and 5min-15 min.

6. The method for preparing a stem cell complex protein preparation for promoting endometrial repair according to claim 5, wherein the stem cell complex protein preparation comprises the following steps: in the step 3.2), the molecular weight cut-off of the first ultrafiltration is 50KD, the molecular weight cut-off of the first concentration is 3KD, and the concentration multiple is 10 times (v/v).

7. The method for preparing a stem cell complex protein preparation for promoting endometrial repair according to claim 6, wherein the stem cell complex protein preparation comprises: in the step 3.3), the molecular weight cut-off of the first concentrated solution after secondary ultrafiltration is 3 KD.

8. The method for preparing a stem cell complex protein preparation for promoting endometrial repair according to claim 7, wherein the stem cell complex protein preparation comprises: in the step 4), the proportion (mass ratio) of each excipient is as follows: 1 to 8 percent of mannitol, 0.5 to 5 percent of dextran, 0.5 to 2 percent of trehalose, and the freeze-drying conditions are as follows: the temperature is-30 ℃ to-80 ℃, the vacuum degree is 30Pa, and the time is 24-72 h.

9. The method for preparing a stem cell complex protein preparation for promoting endometrial repair according to claim 8, wherein the stem cell complex protein preparation comprises: in the step 5.1), the mass of the medical chitosan is 150g-250 g; the use conditions of acetic acid were: the volume is 7ml-10ml, the concentration is O.1M, and the pH is 4.0.

10. The method for preparing a stem cell complex protein preparation for promoting endometrial repair according to claim 9, wherein the stem cell complex protein preparation comprises: in the step 5), the chitosan dissolving conditions are as follows: gradually adding chitosan powder into solvent within 8-20min, and continuously magnetically stirring at room temperature for 1.5-2.5 h to completely dissolve; the dissolving conditions of the beta-sodium glycerophosphate are as follows: dissolving beta-GP with the mass of 0.5g-0.85g in sterile water for injection of 1ml-2ml, and stirring for 4min-8min with shaking; the preparation proportion and conditions of the chitosan temperature-sensitive gel are as follows: 1ml of beta-GP solution is gradually dripped into 9ml of chitosan solution, and the stirring is continued for 5min to 15 min.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method of a stem cell composite protein preparation for promoting endometrial repair.

Background

The endometrium is a layer of mucous membrane covered on the inner layer of the uterine wall, is divided into a functional layer and a basal layer, is a highly reproducible tissue, and dynamically and periodically grows, differentiates, falls off and regenerates in the menstrual cycle. Therefore, when the basal layer is damaged by various factors, the regeneration of uterine epithelium and interstitial cells is disturbed, the formation of new blood vessels is damaged, and finally, the self-repair of the endometrium is difficult to realize. For example, frequent history of uterine cavity operation, infection and partial drug use, when the occurrence of endometrial injury is increased, the endometrium loses the proliferation and repair capability, pathological repair can occur between the front wall and the rear wall of the uterus, fibrous scar and adhesion can be formed, and the symptoms of menoxenia, amenorrhea, infertility, habitual abortion, premature labor, placental abnormality and the like can be clinically shown. The potential for effective treatment is limited by the complexity of the uterine environment and function. Thus, there is an urgent need in gynecology to explore new therapeutic approaches to repair endometrial damage, improve microenvironment and tolerance status. Summarizing current therapeutic strategies, early intervention to reduce scarring, promote repair and regeneration of the damaged endometrium, and thereby achieve restoration of endometrial structure and function is important.

The stem cells have unique biological characteristics and have great application prospects in the field of regenerative medicine treatment. Based on the research of various animal experimental models and clinical experiments, researchers find that stem cells play an important role in repairing endometrial injuries, and possible mechanisms mainly include: 1. the stem cells have the capacity of differentiating into endometrial cells or vascular cells, can supplement the deficient endometrium and promote the tissue repair process by accelerating the formation of new blood vessels in the damaged area; 2. the stem cells have strong paracrine function, can secrete various cytokines such as Vascular Endothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF), basic fibroblast growth factor (bFGF), placenta growth factor (PIGF), platelet derived factor (PDGF) and the like, and can promote the recovery of injured cells, inhibit inflammation and regulate immune response. With the progress of clinical and basic research in recent years, the role of the paracrine mechanism of stem cells in endometrial repair may be greater. Therefore, how to utilize various bioactive factors secreted by stem cells, such as soluble proteins, free nucleic acids, lipids and extracellular vesicles (microparticles, exosomes, etc.), to perform intercellular communication, improve tissue microenvironment, and promote endometrial repair and regeneration has become a hot field of current research.

Through literature search and research, patents CN201910692005.8 and CN201910925566.8 both disclose that the composite repair factor/preparation of human endometrial stem cells is obtained by separating endometrial stem cells from human endometrial tissue, concentrating culture supernatant, freeze-drying, and mixing with a solvent. However, the seed cells used in the invention are endometrial stem cells, the materials of the cells are difficult to obtain, the industrialization is limited, the finally prepared composite repair factor is in a liquid form, and the effective utilization rate of the product in the application process is poor. In addition, the existing stem cell factor products related to the endometrial repair do not specifically induce stem cells to generate a paracrine group for stimulating the repair of endometrial damage, and the obtained stem cell factor products have certain limitation on curative effect.

Therefore, in order to overcome the defects of the prior art, further improvement and promotion of the preparation process are needed, and a preparation method of the stem cell complex protein preparation for promoting endometrial repair, which is suitable for large-scale industrialization and clinical application of patients, is developed.

Disclosure of Invention

In order to solve the technical problems in the background art, the invention provides a preparation method of a stem cell complex protein preparation for promoting endometrial repair, and the preparation method has the advantages of convenience in material obtaining, high cell yield, easiness in control of a production process and low production cost.

The technical solution of the invention is as follows: the invention relates to a preparation method of a stem cell composite protein preparation for promoting endometrial repair, which is characterized by comprising the following steps: the method comprises the following steps:

1) primary extraction and culture of human umbilical cord mesenchymal stem cells:

1.1) extraction of human umbilical cord mesenchymal stem cells: the umbilical cord tissue is obtained from healthy newborn at term, and after tissue sterilization, Wharton's jelly is peeled off and cut into 0.5cm³Centrifugally cleaning tissue blocks with the sizes, adding the tissue blocks into a culture medium, performing conventional culture in an incubator with the temperature of 37 ℃ and the concentration of 5% CO2, replacing the culture medium every 3 days, removing umbilical cord tissues after about 12-14 days, and performing adherent cell culture;

1.2) when the cell fusion degree reaches a certain degree, digesting the cells by using 0.25% pancreatin-EDTA and carrying out passage;

2) specific induction culture of human umbilical cord mesenchymal stem cells:

taking suitable substitute secondary cells, when the cells are cultured to a certain fusion degree, discarding the original culture medium, washing for 3 times by using sterile normal saline, replacing the cells with a serum-free culture medium containing a ferric ion chelating agent Deferoxamine (DFO) for pretreatment culture, and collecting culture supernatant after culture;

3) preparation and concentration of the supernatant of the stem cell-specific protein complex:

3.1) centrifuging the collected culture supernatant to remove cell debris;

3.2) carrying out primary ultrafiltration by using a filter membrane, and then replacing the filter membrane to concentrate the filtrate to obtain primary concentrated solution.

3.3) washing the first concentrated solution twice by using sterile normal saline and performing ultrafiltration again by using a filter membrane, wherein the aim is to completely remove the iron ion chelating agent deferoxamine from the concentrated solution, and the finally obtained concentrated solution is the stem cell concentrated factor and is stored at the temperature of minus 80 ℃;

4) freeze-drying of stem cell concentration factor:

measuring the protein concentration of the obtained stem cell concentrated factor, and adjusting the proportion (mass ratio) of each excipient according to the measurement result; adjusting the concentration of the stem cell concentration factor with sterile water for injection, filtering with a 0.22um filter membrane, and then subpackaging and freeze-drying at a rate of 2ml per branch. Freeze-drying to obtain stem cell concentration factor freeze-dried powder with the concentration of 0.2 mg/branch;

5) preparing the chitosan temperature-sensitive gel:

5.1) preparing chitosan: adding medical chitosan powder into acetic acid, stirring with a magnetic stirrer, and standing at 4 deg.C for storage;

5.2) sodium Beta-glycerophosphate (Beta-Glycerol phosphate, Beta-GP) configuration: dissolving beta-GP in sterile water for injection, shaking and stirring, filtering by a 0.22 mu m filter, preparing a solution, and standing and storing at 4 ℃;

5.3) preparing the chitosan temperature-sensitive gel: taking the prepared chitosan and beta-GP out of the environment of 4 ℃, gradually dripping the beta-GP solution on ice into the chitosan solution, continuously stirring to obtain the chitosan temperature-sensitive gel, subpackaging the chitosan temperature-sensitive gel into penicillin bottles by 3 ml/bottle, and storing at 4 ℃.

Preferably, in the step 1.1), the centrifugation condition is room temperature, the centrifugation is carried out for 4-7min under the condition of the rotation speed of 200g-450g, and the added culture medium after the centrifugation and the cleaning is DF-12 culture medium containing 10% volume fraction of fetal bovine serum.

Preferably, in step 1.2), cell digestion is performed when the degree of cell fusion reaches 70-80%.

Preferably, in the step 2), the cells suitable for generation are from generation P3 to generation P6; the concentration of iron ion chelating agent Deferoxamine (DFO) is 100 μ M-200, and the serum-free medium is MSCXeno-FreeSFM (Corning Cat. No. 88-600-CV), and the culture supernatant is collected after 48h of culture.

Preferably, in step 3.1), the supernatant is collected and centrifuged at 4 ℃ for 5min to 15min and 300g to 500 g.

Preferably, in step 3.2), the molecular weight cut-off of the first ultrafiltration is 50KD, the molecular weight cut-off of the first concentration is 3KD, and the concentration multiple is 10 times (v/v).

Preferably, in step 3.3), the molecular weight cut-off of the first concentrated solution after secondary ultrafiltration is 3 KD.

Preferably, in the step 4), the ratio (mass ratio) of each excipient is: 1 to 8 percent of mannitol, 0.5 to 5 percent of dextran, 0.5 to 2 percent of trehalose, and the freeze-drying conditions are as follows: the temperature is-30 ℃ to-80 ℃, the vacuum degree is 30Pa, and the time is 24-72 h.

Preferably, in the step 5.1), the medical-grade chitosan has the mass of 150g-250 g; the use conditions of acetic acid were: the volume is 7ml-10ml, the concentration is O.1M, and the pH is 4.0.

Preferably, in step 5), the chitosan dissolution conditions are as follows: gradually adding chitosan powder into solvent within 8-20min, and continuously magnetically stirring at room temperature for 1.5-2.5 h to completely dissolve; the dissolving conditions of the beta-sodium glycerophosphate are as follows: dissolving beta-GP with the mass of 0.5g-0.85g in sterile water for injection of 1ml-2ml, and stirring for 4min-8min with shaking; the preparation proportion and conditions of the chitosan temperature-sensitive gel are as follows: 1ml of beta-GP solution is gradually dripped into 9ml of chitosan solution, and the stirring is continued for 5min to 15 min.

The cell complex protein preparation prepared by the invention mainly comprises two parts: a stem cell concentration factor freeze-dried powder and a chitosan temperature-sensitive gel. The method comprises the steps of extracting umbilical cord mesenchymal stem cells, specifically inducing and culturing the umbilical cord mesenchymal stem cells to enable the umbilical cord mesenchymal stem cells to secrete a large amount of cell factors with functions of promoting angiogenesis, cell repair and immunoregulation, concentrating, freeze-drying and the like to obtain the stem cell concentrated factor. Meanwhile, sterile chitosan temperature-sensitive gel is prepared, and the stem cell concentrated factor is uniformly mixed into the chitosan temperature-sensitive gel to obtain the stem cell composite protein preparation, so that the stem cell composite protein preparation can be applied to treatment of endometrial repair. Therefore, compared with the prior art, the invention has the following advantages:

1. the method selects the umbilical cord mesenchymal stem cells as the raw materials for producing the stem cell concentrated solution by using the umbilical cord mesenchymal stem cells, and the umbilical cord mesenchymal stem cells are mainly obtained from the umbilical cord, so the method has the advantages of convenient material obtaining, easy collection and transportation, stable biological characteristics, low immunogenicity, no xenogenic rejection reaction, avoidance of ethical disputes, large cell number and the like. Compared with the endometrium stem cell, the umbilical cord mesenchymal stem cell is more convenient to obtain materials, a large amount of seed cells can be obtained by one-time material obtaining, and the endometrium stem cell is mostly extracted from female menstrual blood, so that the pollution risk in the material obtaining, transporting and culturing processes is large, and the production process control and the cost control are not facilitated. Therefore, the umbilical cord mesenchymal stem cells as seed cells have the advantages of convenient material taking, high cell yield, easy control of the production process, low production cost and the like.

2. One major problem in the application of the paracrine group of stem cells is that, in most cases, the concentration of cytokines and growth factors is too low to be used therapeutically. According to the endometrial injury repair mechanism, the human umbilical cord mesenchymal stem cells are specifically induced and cultured by the iron ion chelating agent Desferrioxamine (DFO), so that the secretion capacity of factors related to angiogenesis of the stem cells is specifically enhanced on one hand, the secretion capacity of cytokines with anti-inflammatory activity of the stem cells is specifically enhanced on the other hand, the microenvironment and microcirculation of endometrium are improved, and the self-repair and defense capacity of the endometrium is enhanced. The economic benefit of this is that the production of large numbers of target-producing stem cell secreting groups can reduce the cost of production and ultimately the cost of treatment for the patient. The iron ion chelating agent Deferoxamine (DFO) mainly plays a role of an anoxia simulation agent in the invention, and compared with other treatment stimulators (such as cobalt chloride), the DFO has high clinical safety and has been clinically applied for more than thirty years; secondly, the DFO can finely adjust the pretreatment through concentration in production; finally, DFO has a low molecular weight and can be removed by washing and concentration without affecting the composition and efficacy of the final product.

3. In order to remove DFO from the final product, the invention removes small molecular DFO by secondary washing and secondary ultrafiltration of an ultrafiltration membrane with the cut-off molecular weight of 3KD, thereby ensuring the purity and the effect of the final product.

4. The solvent used by the stem cell complex protein preparation is temperature-sensitive gel, so the preparation method has the advantages of convenient use, direct introduction into the female external genital tract after being uniformly mixed with the freeze-dried powder, filling of the gel, and promoting the repair and immunoregulation of various anti-inflammatory factors, cytokines and the like on the endometrial cells along with the slow release of the stem cell concentration factors in the temperature-sensitive gel.

Drawings

FIG. 1 is an under-lens view of staining of human umbilical cord mesenchymal stem cells after P2 generation, adipogenic induction for 21d and osteogenic induction for 21d, respectively;

FIG. 2 shows the expression of human umbilical cord mesenchymal stem cells CD34, CD105, CD73 and CD 90;

FIG. 3 shows mRNA expression of VEGF-alpha, ANG1 and IL4 in umbilical cord mesenchymal stem cells after DFO-specific induction;

FIG. 4 shows the protein contents of VEGF-alpha and IL4 in the stem cell factor concentrates.

Detailed Description

The steps of the specific embodiment of the invention are as follows:

1) primary extraction and culture of human umbilical cord mesenchymal stem cells:

1.1) extraction of human umbilical cord mesenchymal stem cells: umbilical cord tissue is obtained from a full-term healthy newborn, and after tissue sterilization, Wharton's jelly is peeled off and cut into tissue blocks with the size of 0.5cm3, and the tissue blocks are centrifuged for 4-7min at the room temperature and the rotating speed of 200g-450g, cleaned and cultured in DF-12 culture medium containing 10% fetal calf serum by volume fraction in a conventional culture box at 37 ℃ and 5% CO 2. Replacing the culture medium every 3 days, removing the umbilical cord tissue after about 12-14 days, and culturing adherent cells;

1.2) cell confluence to 70-80% cells were digested with 0.25% pancreatin-EDTA and passaged.

Detecting the differentiation capacity of the human umbilical cord mesenchymal stem cells:

taking P3 generation cells, digesting conventionally according to 2 × 10⁴And inoculating the cells in a 24-well plate at a density of 70%, changing the culture medium into a fat-forming or osteogenic induction culture medium when the cell fusion reaches the density of 70%, changing the culture medium 1 time every 3d, detecting the bone formation condition by Von Kossa staining after 21d, and detecting the fat formation condition by oil red O.

The experimental results are as follows: referring to fig. 1, under the microscope images of the human umbilical cord mesenchymal stem cells after P2 generation, adipogenic induction for 21d and osteogenic induction for 21d, it can be observed that the cells of P2 generation are fusiform or polygonal, and the morphology of the cells is uniform; after the adipogenic induction is carried out for 21d, a plurality of lipid drop structures appear on cells, the cells can be dyed into red under the action of oil red O, and the cytoplasm can be seen to be filled with the red oil drop structures under the microscope; when the cells are induced to be osteogenic for 21d, the cells are in a multilayer nodular structure, and black calcareous deposits can be seen through Von Kossa staining. The obtained cells have the capacity of adipogenic differentiation and osteogenic differentiation and have the differentiation potential of mesenchymal stem cells.

Detecting a human umbilical cord mesenchymal stem cell surface marker:

after the cells of P3 generation were taken, and digested conventionally, the cells were fractionated into 2X 105 cells per sample, labeled with antibodies CD34, CD105, CD73 and CD90, and then resuspended in 400ul of PBS, and detected by flow cytometry.

The experimental results are as follows: referring to fig. 2, the expression of human umbilical cord mesenchymal stem cells CD34, CD105, CD73 and CD90 is detected, and the expression of CD105, CD73 and CD90 of the obtained cells is positive and the expression of CD34 is negative, which indicates that the isolated cells have a typical mesenchymal stem cell phenotype.

2) Specific induction culture of human umbilical cord mesenchymal stem cells:

taking P3-P6 generation cells, culturing the cells until the fusion degree is 50-70%, discarding the original culture medium, washing for 3 times by sterile normal saline, replacing with a serum-Free culture medium MSC Xeno-Free SFM (Corning Cat. No. 88-600-CV) containing 100-200 μ M iron ion chelating agent Deferoxamine (DFO) for pre-treatment culture, culturing for 48h, and collecting the culture supernatant.

mRNA expression levels of the cells VEGF-alpha, ANG1 and IL4 after the specific induction of the DFO are detected, and mRNA expressions of the cells VEGF-alpha, ANG1 and IL4 after the specific induction of the DFO are respectively detected by a real-time quantitative PCR method.

The experimental results are as follows: referring to fig. 3, mRNA expression of umbilical mesenchymal stem cells VEGF- α, ANG1 and IL4 was specifically induced by DFO. The results indicate that mRNA levels of cellular VEGF- α, ANG1, and IL4 were significantly increased by specific induction pretreatment of DFO. DFO treatment increased expression and secretion of the pro-angiogenic factors VEGF-alpha, ANG1, and the anti-inflammatory factor IL 4.

3) Preparation and concentration of the supernatant of the stem cell-specific protein complex:

3.1) centrifuging the collected culture supernatant at 4 ℃ for 5min-15min under the condition of 300g-500g to remove cell debris;

3.2) carrying out primary ultrafiltration by using a filter membrane with the molecular weight cut-off of 50KD, and then replacing the filter membrane with the molecular weight cut-off of 3KD to concentrate the filtrate, wherein the concentration multiple is 10 times (v/v), so as to obtain a primary concentrated solution;

3.3) washing the first concentrated solution twice by using sterile normal saline and carrying out secondary ultrafiltration by using a filter membrane with the molecular weight cutoff of 3KD, so as to completely remove the iron ion chelating agent deferoxamine from the concentrated solution, and finally obtaining the concentrated solution, namely the stem cell concentrated factor, and storing the concentrated solution at the temperature of minus 80 ℃.

Detecting the protein content of VEGF-alpha and IL4 in the stem cell concentration factor:

the stem cell concentration factor is measured by using a human VEGF-alpha ELISA kit and a human IL4ELISA kit, and the specific operation is carried out according to the instruction.

The experimental results are as follows: referring to FIG. 4, the protein content of VEGF-alpha and IL4 in the stem cell factor concentrates. The results showed that the protein contents of VEGF-alpha and IL4 in the stem cell factor concentrates were 81.35 + -10.73 ng/ml and 1.98 + -0.35 pg/ml, respectively, by specific induction pretreatment with DFO, and were significantly increased compared to the group without DFO pretreatment. The content of pro-angiogenic factor VEGF-alpha and anti-inflammatory factor IL4 protein in the stem cell concentrated factor which can be obtained by stimulation after DFO pretreatment is increased.

Freeze-drying and physicochemical properties of the stem cell concentration factor, and detecting microorganisms:

4) the protein concentration of the obtained stem cell concentrated factor is measured, and the ratio (mass ratio) of the excipients is adjusted according to the measurement result. The proportions of the excipients are as follows: 1 to 8 percent of mannitol, 0.5 to 5 percent of dextran and 0.5 to 2 percent of trehalose. Adjusting the concentration of the stem cell concentration factor to be 0.1mg/ml by sterile water for injection, filtering by a 0.22um filter membrane, and then subpackaging and freeze-drying by 2 ml/branch, wherein the freeze-drying conditions are as follows: the temperature is minus 30 ℃ to 80 ℃, the vacuum degree is 30Pa, and the time is 24 hours to 72 hours. Freeze-drying to obtain the stem cell concentration factor freeze-dried powder with the concentration of 0.2 mg/branch. And (3) detecting the obtained freeze-dried powder according to items such as appearance, redissolution, water content, PH, protein concentration, microorganism, endotoxin and the like.

The experimental results are as follows: the freeze-dried powder is white loose powder with smooth surface. Table 1 shows the physicochemical properties and microbial detection results of the lyophilized powders.

TABLE 1 Freeze-dried powder Experimental results

5) Preparing the chitosan temperature-sensitive gel:

5.1) preparing chitosan: adding 150g-250g medical grade chitosan powder into 9ml acetic acid with concentration of O.1M and pH of 4.0, stirring for 1.5h-2.5h by a magnetic stirrer, preparing a solution, and standing at 4 ℃ for preservation; gradually adding chitosan powder into solvent within 8-20min to prevent agglomeration of the powder, and continuously magnetically stirring at room temperature for 1.5-2.5 h to completely dissolve;

5.2) sodium Beta-glycerophosphate (Beta-Glycerol phosphate, Beta-GP) configuration: dissolving 0.5-0.85 g beta-GP in 1-2 ml sterile water for injection, shaking and stirring for 4-8 min, filtering through a 0.22 mu m filter, and storing the prepared solution at 4 ℃;

5.3) preparing the chitosan temperature-sensitive gel: taking the prepared chitosan and beta-GP out of the environment of 4 ℃, gradually dripping 1ml of beta-GP solution into 9ml of chitosan solution on ice, continuously stirring for 5min-15min to obtain the chitosan temperature-sensitive gel, subpackaging the chitosan temperature-sensitive gel into penicillin bottles in a volume of 3 ml/bottle, and storing at 4 ℃.

The above embodiments are only specific embodiments disclosed in the present invention, but the scope of the present invention is not limited thereto, and the scope of the present invention disclosed in the present invention should be subject to the scope of the claims.