WO2014075593A1

WO2014075593A1 - Mesenchymal stem cell injection, preparation method thereof, and application thereof in preparing drug for treating ulcerative colitis

Info

Publication number: WO2014075593A1
Application number: PCT/CN2013/086838
Authority: WO
Inventors: 黄玉香; 高宏; 王丽; 胡建霞; 张学峰
Original assignee: 贾在美
Priority date: 2012-11-14
Filing date: 2013-11-11
Publication date: 2014-05-22
Also published as: WO2014075593A9; CN102920734A

Abstract

The present invention provides a mesenchymal stem cell injection, a preparation method thereof, and application thereof in preparing a drug for treating ulcerative colitis. The mesenchymal stem cell injection comprises the following ingredients: mesenchymal stem cells of 2×10⁵-1×10⁷/ml; DMSO with the volume ratio being 5-10%; human serum albumin with the mass-to-volume ratio being 1-6%; and 1% of low molecular dextran and a compound electrolyte solution.

Description

Mesenchymal stem cell injection, preparation method thereof and application thereof in preparing medicine for treating ulcerative colitis

The invention belongs to the field of biomedicine, and particularly relates to a mesenchymal stem cell injection solution, a preparation method thereof and application thereof in preparing a medicament for treating ulcerative colitis. Background technique

Ulcerative colitis can occur at any age, but is more common in 20-40 years of age. The causes of the disease in developed countries and cities more than in rural areas are still unclear. It is currently considered to be immune disorders, and genetic and environmental factors contribute to the onset of disease. Studies have found that ulcerative colitis is prone to occur in certain families. About 20% of patients with ulcerative colitis have first-degree relatives (ie, cousins/sisters or sisters) who also have ulcerative colitis, so genetic factors Played a role. In addition to symptoms of the gastrointestinal tract, some patients may experience symptoms in other parts of the body, including: red and itchiness of the eyes; mouth ulcers; joint swelling and pain; skin masses and other injuries; osteoporosis; Patients with a history of 8-10 years with ulcerative colitis have a higher risk of colon cancer.

Ulcerative colitis is different from general inflammatory diseases. The intestinal adhesions of patients are severely damaged. Only patients who have undergone fundamental repair can recover. Traditional surgery and drugs relieve only surface inflammatory reactions. The root cause is that the treatment effect is not satisfactory, and the condition will still recur, which will not satisfy the patients and their families.

Mesenchymal stem cells (MSCs) are important members of the Thousand Cell family and are receiving increasing attention due to their multi-directional differentiation potential, hematopoietic support, and promotion of stem cell implantation and self-replication. MSCs have immunogenicity and unique immunomodulatory effects, which can regulate excessive or weak immune responses in the body and reduce the damage of their own abnormal immune response to their own tissues. Moreover, MSCs can differentiate into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, heart muscle, endothelium, etc. under in vivo or in vitro specific induction conditions, and still have many cells after continuous subculture and cryopreservation. To differentiate potential. Placenta and umbilical cord-derived MSCs have great differentiation potential, strong proliferative capacity, low immunogenicity, and materials Features such as limitations, lack of ethical issues, and ease of industrial preparation make it possible to become the most promising pluripotent stem cell for tissue repair, regulation of immune disorders, and cellular therapy for metabolic diseases.

At present, the treatment of ulcerative colitis mainly solves the symptoms and maintains the asymptomatic state. It cannot fundamentally treat the disease. The patient needs to strictly control the diet for life. In this case, it is still easy to relapse. Abdominal pain, diarrhea and mucus pus and blood are serious. It affects the quality of life of patients, and the side effects of drugs such as hormones and sulfasalazine are 4%. As the disease progresses, the complications of ulcerative colitis gradually appear and worsen, and patients with a longer history are prone to colon cancer. The current drug and surgical treatment can not avoid these situations, adjust the immune disorder in patients with ulcerative colitis, and repair the intestinal segment of the lesion is the basis for the treatment of ulcerative colitis.

The course of ulcerative colitis is difficult to cure. The drug can promote the healing of colonic lesions, relieve symptoms such as diarrhea, rectal blood in the stool and abdominal pain, and eliminate the symptoms and maintain the asymptomatic state, but can not treat the disease from the cause, and some patients suffer from the disease. Wide range of intestines, drug treatment can not alleviate the condition, surgical treatment of the diseased intestine segment must be treated, the surgical effect is acceptable, but the complications of surgery are more, including severe bleeding caused by ulcers, intestinal perforation, toxic megacolon, ulceration Wait, the risk is greater. In view of the current increasing incidence of ulcerative colitis and the lack of special treatments, the present invention intends to fundamentally solve the predicament of long-term medication and recurrent episodes of patients, and treat ulcerative colitis.

Disadvantages of the prior art: At present, the treatment of ulcerative colitis mainly depends on diet control, sulfasalazine and hormones, traditional Chinese medicine enema and other drugs, and surgical treatment is needed when the condition is serious. The comprehensive use of these therapies can temporarily control symptoms such as abdominal pain, diarrhea and pus and bloody stools. It can not effectively correct the immune disorder in patients, and can not fundamentally treat ulcerative colitis and prevent the progression and recurrence of the disease. Moreover, drugs such as sulfasalazine and hormones have serious side effects such as allergic reactions, neutropenia or deficiency, thrombocytopenia and aplastic anemia, hemolytic anemia and hemoglobinuria, and liver damage. Surgery can partially cure the disease, but postoperative complications are more serious, such as: adhesive intestinal obstruction, anastomotic bleeding, paralysis, etc., seriously affecting the quality of life of patients. Summary of the invention

In view of the above-mentioned drawbacks of the prior art ulcerative colitis, the present invention provides a mesenchymal stem cell injection, a preparation method thereof and use thereof for the preparation of a medicament for treating ulcerative colitis. The injection can fundamentally regulate the immune disorder of patients with ulcerative colitis, prevent the destruction of intestinal tissues by autoimmune disorders; promote the repair of the intestinal lesions and the healing of ulcers, thereby freeing patients from abdominal pain and diarrhea, hormones The toxic side effects caused by drugs, as well as serious complications caused by poor disease control.

In order to achieve the above object, the present invention is implemented by the following technical solutions:

A mesenchymal dry fine ^ ^ injection, which comprises the following components:

Mesenchymal stem cells in a concentration of 2 X 10 ⁵ - 1 X 10 ⁷ /ml;

a clinical grade DMSO with a volume ratio of 5-10%;

Human albumin with a mass to volume ratio of 1-6%;

a low molecular weight dextran having a mass to volume ratio of 1%;

The balance is a compound electrolyte solution.

Further improvements to the above technical solution: The mesenchymal stem cells are derived from a human umbilical cord and/or a placenta. A further improvement of the above technical solution: the viability of the mesenchymal stem cells is maintained above 85%. The present invention also provides a method for preparing the mesenchymal stem cell injection, which comprises the preparation of umbilical cord mesenchymal stem cells and the preparation of placental mesenchymal stem cells.

The invention also provides the use of the mesenchymal stem cell injection for the preparation of a medicament for treating ulcerative colitis.

Further improvement of the above technical solution: The mesenchymal stem cell injection is used in an amount of 1 X 10 ⁵ - 1 X 10 ⁸ /ml mesenchymal stem cells.

Compared with the prior art, the advantages and positive effects of the present invention are:

1. The present invention selects mesenchymal stem cells derived from human umbilical cord and placenta to prepare mesenchymal stem cells. The mesenchymal stem cells have a large yield, and the preparation system is easy to control and easy to industrialize.

2. The composition of mesenchymal stem cell injection consists of human-filled shield stem cells, clinical grade DMSO, human serum albumin, low molecular weight dextran, and boehm force (combined electrolyte solution). This injection can be directly After program-controlled cooling and cryopreservation, it can be directly used for clinical injection after resuscitation.

3. The present invention utilizes human mesenchymal cell injection to correct immune disorders in patients with ulcerative colitis, treats diseases from the etiology, prevents autoimmune disorders from continuing to destroy the intestinal tract, and secretes many cell growth factors and repair factors. It promotes the healing of ulcers in the affected segment of the intestine, thereby achieving the purpose of fundamentally treating ulcerative colitis. The invention can reverse the course of ulcerative colitis, prevent the occurrence of complications, help the patient to get rid of the influence of abdominal pain and diarrhea on life and the side effects caused by drugs, and thoroughly treat ulcerative colitis.

Other features and advantages of the present invention will become apparent from the Detailed Description of the Drawing. DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a photograph showing the appearance of human umbilical cord mesenchymal cells in the present invention.

Fig. 2 is a graph showing the comparison of changes in the colon tissue of rats in the present invention.

Fig. 3 is a graph showing the comparison of pathological changes after treatment of rat colon tissue in the present invention.

Fig. 4 is a graph showing changes in serum TNF-α levels in rats after treatment in the present invention. detailed description

The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings and specific embodiments. Example 1

I. Preparation of umbilical cord mesenchymal stem cells

1. Fresh full-term healthy fetal umbilical cord, rinsed with PBS buffer containing 100 kU/L penicillin and 100 mg/L streptomycin;

2. Cut a 5-10 cm long umbilical cord, cut the umbilical cord into 2 cm long sections, and wash repeatedly with PBS buffer; the PBS buffer contains 100 kU/L penicillin and 100 mg/L streptomycin;

3. Cut the umbilical tissue block into 2-5 mm ³ small pieces; wash it in L-DMEM medium, centrifuge at 500-800 g for 5 minutes, discard the supernatant; 4. Add the tissue block and the medium to the medium at a volume ratio of 2.5-3:1, mix the tissue pieces, inoculate the cells into the culture dish, and incubate the culture medium; the medium is l-10ng/ml Basic fibroblast growth factor (bFGF) and serum-free medium for MSC;

5. Change the medium every 3 days, and pass through the cells when the cells reach 80% confluence in 8-9 days; the subculture medium is the serum-free medium for MSC.

Second, the preparation of placental mesenchymal stem cells

1. Rinse the placenta with PBS buffer containing 100 kU/L penicillin, 100 mg/L streptomycin and 50 u/ml heparin sodium, and slowly peel the fetal amniotic membrane along the edge of the placenta;

2. Rinse the placenta source amniotic membrane again, and cut the amniotic membrane into l-5mm ³ small pieces;

3. The amniotic tissue block is mixed with DMEM medium containing penicillin and streptomycin, and centrifuged at 700-950 g for 10 min;

4. Discard the supernatant, each tube was mixed with 0.25% trypsin in DMEM medium and digested at 37 ° C for 10 min, centrifuged at 700-950 g for 10 min;

5. Discard the supernatant, add complete medium to each tube, and centrifuge at 2200 rpm for 10 min; the complete medium is MSC serum-free medium containing 100 kU/L penicillin + 100 mg/L streptomycin;

6. Discard the supernatant, inoculate the amniotic tissue block in the culture medium, add the complete medium, and culture in the incubator; perform half-time change every 3 days;

7. After 10-12 days, there is cell adherent growth. After the cell clone is formed, the tissue block is discarded, and the whole culture medium is added for cultivation;

8. To 15-17 days, the cell clone was fused to 80-90% for cell passage.

3. Preparation of mesenchymal stem cell injection

The injection is prepared in proportion to the following ingredients:

1, derived from human umbilical cord and / or placental mesenchymal stem cells, the number of mesenchymal stem cells per ml of injection is 2x l0 ^{5 -} lx l0 ⁷ ;

2, volume ratio of 5-10% DMSO (clinical grade);

3. Human albumin with a mass to volume ratio of 1-6%; 4, mass to volume ratio of 1% low molecular dextran

5. The balance is the pulse force (combined electrolyte solution).

Clinical grade DMSO is non-toxic to cells and can better protect cells. It does not need to be rinsed after cell rejuvenation. It can be directly used for clinical injection and is safe and safe for patients.

Human serum albumin is a commonly used clinical injection, which can provide nutrition for cells and facilitate cell metabolism.

The addition of 1% low molecular weight dextran ensures that the cells maintain a good cell dispersion state during storage, which reduces the adhesion between cells and the cell adhesion to the container wall, and reduces the intravascular cell formation that may occur during clinical cell infusion. The risk of embolization of the mass also reduces the cell loss caused by cell aggregation and filtration by the infusion filter.

Bourgeois (complex electrolyte solution) can maintain the osmotic pressure of cells and facilitate cell survival. The injection component is a clinically used electrolyte solution, which is convenient for clinical infusion.

The injection can be stored stably at -196 °C. When thawed and resuscitated, it can be directly infused into the patient. The cells remain in a single cell suspension state, and the cell viability remains above 85% without causing uncomfortable reactions.

This kind of injection is very beneficial to the preservation, transportation and survival of mesenchymal stem cells, and the clinical infusion is safe. The cells can maintain high vitality in this injection for a long time, and it is easy to transport without being restricted by the time of use. The problem of long-term cell transport affecting cell viability when used by patients in different places.

The specific preparation process of mesenchymal stem cell injection is as follows: For example, 100 ml of stem cell injection is prepared, which is prepared by human mesenchymal stem cells, clinical grade DMSO 5 ml, human albumin stock solution (stock solution mass concentration ratio of 20%) 10 mL low. Molecular dextran 1ml (base solution concentration 100%) and Bobme force (combined electrolyte solution) 84ml. The mass to volume ratios of the present invention all represent the ratio of mass (g) to volume (ml). The number of mesenchymal cells per ml of injection is 2χ 10 ⁵ -1 χ 10 ⁷ . Mesenchymal stem cell injection can maintain a single cell suspension at -196 ° C ambient temperature, and the cell viability remains above 85% after resuscitation, which can be directly used for clinical injection.

Safety and effectiveness of mesenchymal stem cell injection in rats with ulcerative colitis 45 healthy SD rats, 6-8 weeks old, were randomly divided into 3 groups, 15 in each group. Adoption A model of ulcerative colitis was prepared by the complication method. Rats in groups A and B were injected with 0.4 ml of emulsion in the groin and toe at 1 and 14 days after the experiment. Fasting after immunization (not allowed to drink water) 24h, 3% chloral hydrate was intraperitoneally injected into the anesthesia, and 0.6ml liquid (100 mg/kg TNBS ten 50% ethanol) was pushed through the anus. After modeling for 24 hours, rats in group A were injected with lml human mesenchymal stem cell injection (containing 5×10 ⁶ cells, electron micrograph shown in Figure 1). Group B rats were injected with 1 ml PBSo C group via tail vein. SD rat.

Rats were observed daily for signs and symptoms after the start of the experiment. Five rats were anesthetized on the 3rd, 7th, and 14th day after treatment with mesenchymal stem cells. The colon was dissected and the colonic injury was observed. The pathological changes of the colon were observed under a microscope.

Results: There was no acute shock or death in the rats throughout the experiment.

1. Observation of living conditions of rats: One day after model establishment, rats in group A and group B began to develop anorexia, laziness, increased stool frequency, soft stool or loose stool. After 3 days, the symptoms reached a peak, and then the symptoms began to relieve. The remission rate of group A was faster than that of group B. At the end of the experiment, group A had no diarrhea and the activity was normal. There were no abnormal symptoms in group C.

2. Colon tissue changes: 3 days after treatment, both groups A and B showed thickening of the colonic wall, congestion and edema, and ulcers in a large area in some areas; 7 days after treatment, group A was slightly relieved of congestion and edema The range of ulcers was also reduced. There was no significant change in the lesions in group B. At 14 days after treatment, only mild hyperemia and edema were seen in group A, no ulcers were seen, and lesions in group B were slightly relieved. The experimental findings are shown in Figure 2, left. For: group A rat mesenchymal stem cells 3 days after treatment, colon tissue, colonic intestinal wall thickening, congestion and edema, partial ulcers in a large area; the middle picture is: group A rat mesenchymal stem cells after treatment In the 14-day colon tissue, the lesions were significantly relieved and the ulcers healed. The right picture shows: The colon tissue of the rats in group B was treated 14 days after treatment, and there were still obvious hyperemia and edema and macroscopic ulcers.

3. Colonic pathological changes: 3 days after cell treatment, a group of colonic pathological sections showed a wide range of epithelial defects under HE staining, inflammatory cell infiltration in mucosa and submucosa, and pathological changes in group B were similar to group A; In the last 7 days, the extent of epithelial defects in group A was reduced, and a large number of inflammatory cells infiltrated in the mucosa and submucosa. The lesions in group B did not change significantly. By 14 days, the mucosal structure of group A was almost intact, and only a small amount of inflammatory cells infiltrated. As shown in Figure 3, the left picture is: Group A rat mesenchymal stem cells Colon tissue pathology 3 days after treatment, a large range of epithelial defects were observed under the microscope, and inflammatory cells infiltrated in the mucosa and submucosa; the middle picture is: colonic histopathology of group A rat mesenchymal stem cells 14 days after treatment, visible under the microscope The mucosal structure is basically intact, only a small amount of inflammatory cells infiltrate; the right picture shows: Colon tissue pathology of group B rats 14 days after treatment, there is still a wide range of epithelial defects under the microscope, inflammatory cell infiltration in the mucosa and submucosa.

4. Changes of immune index in rats: 3 days after cell treatment, serum TNF-α levels in group A were significantly higher than those in control group. Group B changes were similar to group A; 7 days after treatment, serum TNF in group A rats The content of -a decreased, and the lesions of group B did not change significantly. By 14 days, the serum TNF-a content of group A was significantly lower than that of group B. The experimental results are shown in Figure 4. 14 days after treatment, group A rats Serum TNF-a levels were significantly lower than those in group B (P<0.05).

Conclusion: Human mesenchymal stem cell injection is safe and feasible in the treatment of ulcerative colitis rats. After treatment with mesenchymal stem cells, the living conditions, symptoms and signs, colon pathology and immune disorder of the rats are obviously improved. effective.

Example 2

For example, 100ml stem cell injection is prepared, which consists of human mesenchymal stem cells, clinical grade DMSO 10ml, human serum albumin stock solution (20% stock solution) 10ml, low molecular weight dextran 1ml and Bomagin (complex electrolyte solution) 79ml. . The number of mesenchymal stem cells per ml of injection is 2 X 10 ⁵ - 1 X 10 ⁷ . Mesenchymal stem cell injection can maintain a single cell suspension at -196 °C ambient temperature, and the cell viability remains above 85% after resuscitation, which can be directly used for clinical injection. Example 3 Culture and detection of umbilical cord mesenchymal stem cells The umbilical cord mesenchymal stem cells prepared in Example 1 were used for amplification. Cell culture amplification was inoculated at a density of 1.0-1.2 X lOVcm ² , and completely serum-free medium was added. After the cell fusion degree was 80-90%, the cells were collected by room temperature digestion with 0.05% trypsin (without EDTA). The cells should not be over-fused, otherwise growth inhibition will not only occur, but also stem cells will spontaneously differentiate, which will seriously affect the cell growth state after passage. Immunophenotypic determination of umbilical cord mesenchymal stem cells: 1 X 10 ⁶ P1, P6 cell numbers were collected, mouse anti-human PE-IgG1, FITC-IgGl isotype control were added, and PE, FITC-labeled mouse anti-human antibody was added. Detection of CD 34, CD 45 (hematopoietic cell marker), CD3 1 (endothelial cell-specific antigenic marker), CD14 (mononuclear macrophage surface marker), CD 90, CD 44, CD 105 (mesenchymal antigen marker), Immunophenotypes such as HLA-DR (transplantation immune rejection related antigen). Cell culture results and detection: Under the microscope, the cells adhered to the wall, and the morphology should be fusiform, with high refractive index. The cells are evenly arranged and arranged in a swirling shape. The number of primary cells harvested is > 1 10 ⁷ ; cell viability (Taiwan Blue staining): The cell viability rate is 90% before cryopreservation and 85% after cryopreservation. The cells are continuously transferred to the 6th generation. The cells are stable in shape, fusiform, evenly distributed, and arranged neatly. The cell proliferation rate is stable after continuous transmission to the 6th generation. The phenotypes are consistent with the MSC identification criteria (CD73, CD 105, CD44 or CD90 positive, the positive rate is not less than 95%; CD3 1, CD34, CD45, HLA-DR is negative, the positive rate should not be higher than 2%. Cell cycle detection: 70-80% of cells are in the G0G1 phase of the cell cycle; P l and P6 cells have multi-directional differentiation ability; karyotype analysis is normal; the total number of expanded cells in 5-6 generations can reach 10 ^1G ~10 ^U. The partial flow test results are shown in Figure 5. Among them, the average number of primary culture days is 13 days, and the total number of harvested cells can reach 1.6 X 10 ⁷ . After 6 consecutive generations, the cells grow well, showing a uniform small spindle shape, and the swirling arrangement is neat. The primary cells are shown in Figure 6 (p. Photographs of 9 and 13 days), cells of the 6th generation are shown in Figure 7 (photos on days 1 and 4). Example 4 Stem cell therapy was performed on a patient suffering from ulcerative colitis using the stem cell injection of Example 2. Treatment: Intravenous injection of mesenchymal stem cells 50ml, containing 3 X 10 ⁷ cells, 1 week after the intervention of mesenteric artery injection of mesenchymal stem cells 10ml, the number of cells containing 1.5 χ 10 ⁷ . Case 1: Patient A, female, 36 years old, mucus bloody stool for more than 4 years, stool 2-3 times, soft, not formed. With general malaise, paroxysmal pain in the left lower abdomen, and reduced abdominal pain after the stool. Colonoscopy: (rectal, sigmoid colon) diffuse hyperemia, partial superficial erosion, scattered in patchy ulcers. Pathology: (rectal, sigmoid colon) mucosal severe chronic active inflammation with shallow ulcers and crypt abscess formation. See Figure 8. 3 months after treatment, there is a small amount of mucus in the stool, no stool with blood, no abdominal pain, abdominal distension and other abdominal discomfort, stool 1-2 times a day, soft, forming. In order to consolidate the curative effect, the patient performed stem cell implantation again 14 weeks after treatment. After treatment, the symptoms basically disappeared, and the colonoscopy was almost normal. See Figure 9. Case 2: Patient Wang Moumou, male, 50 years old, mucus pus and bloody stool for more than 3 years, accompanied by abdominal pain and abdominal distension. The patient had 5-6 times of stool on admission, with pus and blood, and more pus. Colonoscopy: (rectal) diffuse congestive edema, superficial erosion, surface exudation, and scattered in the formation of shallow ulcers. Pathology: The rectal mucosa is characterized by moderate to severe chronic active inflammation with ulcers and crypt abscess formation. See Figure 10. After two months of treatment, the patient's symptoms basically disappeared, there was no pus in the stool, abdominal pain, abdominal distension and other abdominal discomfort. The stool was 1-2 times a day, soft and shaped. Colonoscopy: Mild diffuse hyperemia of the rectal mucosa. Pathology: mild chronic inflammation of the rectal mucosa. See Figure 11. After 6 months of treatment, the clinical symptoms disappeared, there was no pus in the stool, abdominal discomfort such as abdominal pain and bloating, and stools were taken 1-2 times, soft and shaped.

The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to be limiting; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still The technical solutions described herein are modified or equivalently replaced with some of the technical features; and such modifications or substitutions do not depart from the spirit and scope of the technical solutions claimed in the present invention.

Claims

Rights request

1. A mesenchymal cell injection, characterized in that it includes the following components:

Mesenchymal stem cells with a content of 2χ 10 ⁵ -1 χ 10 ⁷ cells/ml;

5-10% clinical grade DMSO by volume;

Human albumin with a mass-to-volume ratio of 1-6%;

Low molecular weight dextran with a mass/volume ratio of 1%;

The remainder is compound electrolyte solution.

2. The mesenchymal stem cell injection according to claim 1, characterized in that: the mesenchymal stem cells are derived from human umbilical cord and/or placenta.

3. The mesenchymal stem cell injection according to claim 1, characterized in that: the viability of the mesenchymal stem cells is maintained at more than 85%.

4. The preparation method of mesenchymal stem cell injection according to claim 1, characterized in that the preparation volume ratio is DMSO of 5-10%, the mass-volume ratio of human serum albumin is 1-6%, and the mass-volume ratio is 1-6%. It is a 1% low molecular weight dextran and compound electrolyte solution, and the mesenchymal stem cells are resuspended in the above solution to make a single cell suspension, so that the number of stem cells is 2 × 10 ⁵ - 1 × 10 ⁷ /ml.

5. The preparation method of mesenchymal stem cell injection according to claim 4, characterized in that the preparation of umbilical cord mesenchymal stem cells includes the following steps:

(1). Take fresh full-term healthy fetal umbilical cord and wash it with PBS buffer containing 100 kU/L penicillin and 100 mg/L streptomycin;

(2). Cut the 5-10cm long umbilical cord, cut the umbilical cord into 2cm long segments, and rinse repeatedly with the PBS buffer;

(3). Cut the umbilical cord tissue pieces into ^small pieces of 2-5 mm; add L-DMEM culture medium for washing, centrifuge at 500-800g for 5 minutes, and discard the supernatant;

(4). Add the tissue block and culture medium to the medium at a volume ratio of 2.5-3:1, mix the tissue block, and inoculate it into cell culture for culture. The medium contains 1-10ng/ml alkaline ingredients. fibroblast growth factor Serum-free medium specifically for MSC;

(5). Change the medium every 3 days until the cells reach about 80% confluence on 8-9 days. The subculture medium is MSC-specific serum-free medium.

6. The method for preparing mesenchymal stem cell injection according to claim 4, characterized in that the preparation of placental mesenchymal stem cells includes the following steps:

(1). Rinse the placenta with PBS buffer containing 100 kU/L penicillin, 100 mg/L streptomycin and 50u/ml heparin sodium, and slowly peel off the amniotic membrane on the fetal face along the edge of the placenta;

(2). Rinse the placenta-derived amniotic membrane again and cut the amniotic membrane into ^1-5mm pieces;

(3). Mix the amniotic tissue pieces with DMEM culture medium containing penicillin and streptomycin, and centrifuge at 700-950g for 10 minutes;

(4). Discard the supernatant, add DMEM containing 0.25% trypsin by volume to each tube, digest for 10 minutes at 37°C, and centrifuge at 700-950g for 10 minutes;

(5). Discard the supernatant, add complete culture medium to each tube, and centrifuge for 10 minutes under 00rpm conditions; the complete culture medium is MSC serum-free culture medium containing 100 kU/L penicillin + 100 mg/L streptomycin;

(6). Discard the supernatant, inoculate the amniotic tissue pieces in a petri dish, add complete culture medium, and culture in an incubator; change half of the medium every 3 days;

(7). After 10-12 days, cells will grow adherently and cell clones are formed. Discard the tissue block and add complete culture medium for culture;

(8). On days 15-17, cell clone confluence reaches 80-90%, and cells are passaged.

7. Application of the mesenchymal stem cell injection according to claim 1 in the preparation of drugs for the treatment of ulcerative colitis.

8. The application of the mesenchymal stem cell injection according to claim 7 in the preparation of drugs for the treatment of ulcerative colitis, characterized in that: the dosage of the mesenchymal stem cell injection contains 1 χ10 ⁶ -1 χ10 ⁸ mesenchymal stem cells/ml.