WO2014075593A9

WO2014075593A9 - Mesenchymal stem cell injection, preparation method thereof, and application thereof in preparing drug for treating ulcerative colitis

Info

Publication number: WO2014075593A9
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: 2014-07-03
Also published as: WO2014075593A1; 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 and 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 and 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. Developed countries and cities are more than rural areas. The cause of the disease is still unclear. It is currently considered to be an immune disorder, and the combination of genetic and environmental factors leads to disease. Studies have found that ulcerative colitis is prone to occur in certain families, and 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 patient's intestinal mucosa is seriously damaged. Only patients who have undergone fundamental repair can recover. Traditionally, surgery and medicine alleviate only the surface inflammatory reaction, and the symptoms are not cured. Therefore, the treatment effect is not satisfactory, and the condition will still recur, and the patient and family members cannot be satisfied.

Mesenchymal stem cells (MSCs) are important members of the stem 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 low immunogenicity and unique immunoregulatory effects, which can regulate excessive or weak immune response in vivo 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 successively subculture and cryopreservation correction pages ( Rule 91) ISA/CN Still have multi-directional differentiation potential. Placenta and umbilical cord-derived MSCs have the characteristics of large differentiation potential, strong proliferative ability, low immunogenicity, convenient materials, no moral and ethical problems, and easy industrial preparation, making it possible to regulate tissue repair and immune disorders. And pluripotent stem cells with the most clinical application prospects for metabolic disease cell therapy.

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 very large. 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 fact that the incidence of ulcerative colitis is increasing year by year and there is no specific and simple treatment method, the present invention intends to fundamentally solve the dilemma 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 surgery is required 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 correct (Article 91) ISA/CN More and 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 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 achieved by the following technical solutions:

A mesenchymal stem cell injection comprising the following components:

Mesenchymal stem cells with a content of 2x l0 ⁵ -l xl0 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 mesenchymal stem cell activity is maintained above 85%.

The present invention also provides a method for preparing the mesenchymal stem cell injection, which comprises preparing a umbilical cord mesenchymal stem cell and preparing a placental mesenchymal stem cell.

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

Further improvement to the above technical solution: the amount of the mesenchymal stem cell injection is corrected (page 91) ISA/CN 1 x 10 ⁶ - 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 a mesenchymal stem cell injection using mesenchymal stem cells derived from a human umbilical cord and a placenta. The mesenchymal stem cells have a large yield, and the preparation system is easy to control and easy to industrialize.

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

3. The present invention utilizes human mesenchymal stem 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. Promote the healing of the ulcer of the diseased intestine, so as to achieve 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 impact of abdominal pain and diarrhea on life and the side effects of 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 micrograph of human umbilical cord mesenchymal stem cells in the present invention - Fig. 2 is a comparison diagram of changes in rat colon tissue after treatment 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.

Figure 5 is a partial flow test result of cells in the present invention.

Fig. 6 is a photograph of Days 9 and 13 of the primary cells of the present invention.

Figure 7 is a photograph of Days 1 and 4 of the 6th generation cells of the present invention.

Figure 8 is a colonoscopy of a case 1 patient in the present invention. Correction page (Article 91) ISA/CN Fig. 9 is a view showing the colonoscopy of the case 1 patient in the present invention 3 months after the treatment.

Figure 10 is a colonoscopy of a case 2 patient in the present invention.

Figure 11 is a colonoscopy of a patient in Case 2 of the present invention after two months of treatment. detailed description

The technical solutions of the present invention will be 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 rinse 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; add to L-DMEM medium, centrifuge at 500-800 g for 5 minutes, discard the supernatant;

4. The tissue block and the culture medium are added to the medium at a ratio of 2.5-3:1 by volume, and the tissue block is mixed, inoculated into the cell culture, and cultured in an incubator; the medium is 1.-10 ng/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; Correction page (Rule 91) ISA/CN 4. Discard the supernatant, each tube was digested with DMEM medium containing 0.25% trypsin for 10 min at 37 ° C, and 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, add the complete medium, and incubate in the incubator; half a change of liquid 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 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 thousand cell injection

The injection is prepared in proportion to the following ingredients:

1. From human umbilical cord and/or placenta mesenchymal stem cells, the number of mesenchymal stem cells per ml of injection is 2χ 10 ⁵ -1 χ 10 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 resuscitation. It can be directly used for clinical injection and is safe and safe for patients.

Human serum albumin is a clinically used injection that provides nutrients to cells and facilitates 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. Correction page (Article 91) ISA/CN The injection component is a common clinical electrolyte solution, which is convenient for clinical infusion.

The injection can be stably stored at a temperature of -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%, and the patient does not cause 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: preparation of 100ml thousand cell injection, the injection consists of human mesenchymal stem cells, clinical grade DMSO 5ml, human albumin stock solution (stock solution mass concentration ratio of 20%) 10ml , low molecular weight right-handed glycine liver lml (stock solution concentration of 100%) and Bomai force (combined electrolyte solution) 84ml composition. The mass to volume ratios of the present invention all represent the ratio of mass (g) to volume (ml). The number of mesenchymal stem 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 efficacy of mesenchymal stem cell injection in the treatment of ulcerative colitis in rats

Forty-five healthy and clean SD rats, 6-8 weeks old, were randomly divided into .3 groups, 15 in each group. An ulcerative colitis model was prepared by immunocomplexation. A and B rats were injected with 0.4 ml of emulsion in the groin and toe at 1 and 14 days after the experiment. Fasted after immunization (not allowed to drink water) 24h, 3% chloral hydrate was intraperitoneally injected into the anesthesia, and 0.6ml liquid (100mg/kg TNBS + 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>< ¹⁰⁶ cells, electron micrograph shown in Figure 1), and rats in group B were injected with lml PBS via tail vein. Group C was a normal 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 colon injury was observed. On the correction page (Article 91) ISA/CN 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 in rats: One day after modeling, rats in groups A and B began to develop anorexia, laziness, increased stool frequency, soft stools or loose stools. 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. Group C did not show any abnormal symptoms.

2. Changes in colonic tissue: 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 7 days after treatment, group A was slightly relieved of congestion and edema. Also reduced, there was no significant change in group B lesions; 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 results are shown in Figure 2. The left picture is: Group A Three days after treatment with murine mesenchymal stem cells, the colonic wall was thickened, congested and edema, and ulcers were seen in some areas. The middle picture shows: colon tissue of group A rat mesenchymal stem cells after 14 days of treatment. The lesions were significantly relieved and the ulcers healed; the right picture shows: Colon tissue in group B after 14 days of treatment, there are still obvious congestion and edema and macroscopic ulcers.

3. Histopathological changes of colon tissue: 3 days after cell treatment, a large range of epithelial defects were observed under HE staining microscope in group A, and inflammatory cells infiltrated in mucosa and submucosa. Group B pathological changes were similar to group A; In the 7th day, 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 panel is: colonic histopathology 3 days after treatment of group A rat mesenchymal stem cells, a large range of epithelial defects can be seen under the microscope, and inflammatory cells infiltrate in the mucosa and submucosa; Colonic histopathology was observed 14 days after rat mesenchymal stem cells treatment. Microscopically, the mucosal structure was almost intact, and only a small amount of inflammatory cells were infiltrated. The right picture shows: Colon tissue pathology of group B rats 14 days after treatment, still large under microscope A range of epithelial defects, inflammatory cell infiltration in the mucosa and submucosa.

4. Changes in 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. Seven days after treatment, group A rats were corrected for serum. Page (Rules 91) ISA/CN The content of TNF-α was decreased, and the lesions in group B were not significantly improved. By 14 days, the serum TNF-a content in group A was significantly lower than that in group B. The experimental results are shown in Figure 4. 14 days after treatment, group A was large. The serum TNF-α level in rats was significantly lower than that 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 Bobme force (combined electrolyte solution) 79ml. . The number of mesenchymal stem 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. 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 10 ⁴ /cm 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. Immunophenotyping of umbilical cord mesenchymal stem cells: 1 X 10 ⁶ Pl, P6 cell numbers were collected, mouse anti-human PE-IgG l, FITC-IgGl isotype control were added, and PE, FITC-labeled mouse anti-human flow antibody was added. , detection of CD 34, CD 45 (hematopoietic cell marker), CD31 (endothelial cell-specific antigenic marker), CD14 (mononuclear macrophage surface marker), CD 90, CD 44, CD105 (mesenchymal antigen marker), HLA -DR (transplant free

Correction page (Article 91) ISA/CN Immunophenotype such as epidemic rejection related antigens. 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 X 10 ⁷ ; cell viability (Taiwan Panlan staining: 90% survival rate before cryopreservation, cell viability after cryopreservation 85%; continuous passage to 6th generation, cell morphology is stable, fusiform, distributed, and arranged neatly; continuous transmission to 6th generation cell proliferation The phenotype is consistent with the MSC identification criteria (CD73, CD 105, CD44 or CD90 positive, the positive rate is not less than 95%; CD31, 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; Pl and P6 cells have multi-directional differentiation ability; karyotype analysis is normal; the total number of consecutive 5-6 generation cells can reach lO^ lO^ Partial flow detection results are shown in Figure 5. Among them, the average average culture days is 13 days, and the total number of harvested cells can reach 1.6 X 10 ⁷ . After 6 consecutive passages, the cells grow well, showing a uniform small spindle shape, arranged in a swirling manner, and the primary cells are shown in Figure 6 (p. Photographs of 9 and 13 days), the 6th generation cells 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 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) Mucosal severe chronic active inflammation with shallow ulcers and crypt abscess formation. See Figure 8.

Correction page (Article 91) ISA/CN 3 months after treatment, there is a small amount of mucus in the stool, no blood in the stool. There is no abdominal discomfort such as abdominal pain and abdominal distension. The stool is 1-2 times a day, soft and shaped. 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 hyperemia and edema of the mucous membranes, superficial erosion, surface exudation, and scattered shallow ulcer formation. 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.

Correction page (Article 91) ISA/CN

Claims

Rights request

A mesenchymal thousand cell injection, characterized in that it comprises the following components:

Mesenchymal stem cells with a content of 2χ 10 ⁵ -1 χ 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.

The mesenchymal stem cell injection according to claim 1, wherein the mesenchymal stem cells are derived from a human umbilical cord and/or a placenta.

The mesenchymal stem cell injection according to claim 1, wherein the activity of the mesenchymal stem cells is maintained at 85% or more.

The method for preparing a mesenchymal stem cell injection according to claim 1, characterized in that a volume ratio of 5-10% of DMSO, a human to albumin having a mass to volume ratio of 1-6%, and a mass to volume ratio are prepared. The monocellular suspension was prepared by resuspending the mesenchymal stem cells in the above solution as 1% low molecular dextran and a compound electrolyte solution, so that the number of stem cells was 2 X 10 ⁵ - 1 X 10 ⁷ /ml.

The method for preparing a mesenchymal stem cell injection according to claim 4, wherein the preparation of the umbilical cord mesenchymal stem cells comprises the following steps:

(1) Take a fresh full-term healthy fetal umbilical cord and rinse 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 rinse repeatedly with the PBS buffer;

(3) The umbilical tissue block was cut into 2-5 mm ³ small pieces; washed in L-DMEM medium, centrifuged at 500-800 g for 5 minutes, and the supernatant was discarded;

(4) The tissue block and the culture medium are added to the medium at a volume ratio of 2.5-3:1, the tissue pieces are mixed, and inoculated into the cell culture, the medium is alkaline-containing l-10 ng/ml. Fibroblast growth factor Serum-free medium for MSC;

(5). Change the medium every 3 days, and pass the passage when the cells reach about 80% confluence in 8-9 days. The subculture medium is the serum-free medium for MSC.

The method for preparing a mesenchymal stem cell injection according to claim 4, wherein the preparation of the placental mesenchymal stem cells comprises the following steps:

(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 is added with DMEM medium containing 0.25% trypsin and digested at 37 ° C for 10 min, centrifuged at 700-950 g for 10 min;

(5). The supernatant was discarded, and after each tube was added with complete medium, it was centrifuged at OO rpm for 10 min; the complete medium was 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 dish, 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 medium is added for cultivation;

(8). By 15-17 days, the cell clone was fused to 80-90%, and cell passage was performed.

The use of the mesenchymal stem cell injection according to claim 1 for the preparation of a medicament for treating ulcerative colitis.

The use of the mesenchymal stem cell injection according to claim 7, wherein the mesenchymal stem cell injection comprises 1 χ ^{10 6} -1 χ ^{10 8 .} Mesen/ml mesenchymal stem cells.