CN116064382A - Cell preparation for treating anal fistula complicated with Crohn disease - Google Patents
Cell preparation for treating anal fistula complicated with Crohn disease Download PDFInfo
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- CN116064382A CN116064382A CN202111282051.4A CN202111282051A CN116064382A CN 116064382 A CN116064382 A CN 116064382A CN 202111282051 A CN202111282051 A CN 202111282051A CN 116064382 A CN116064382 A CN 116064382A
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Abstract
The invention belongs to the technical field of biological medicines, relates to a human umbilical cord mesenchymal stem cell preparation, and in particular relates to a CD123 positive human umbilical cord mesenchymal stem cell preparation, a preparation method thereof and application thereof in treating Crohn's disease complicated anal fistula. The cell preparation provided by the invention can fundamentally improve the inflammation of patients with the complicated anal fistula of Crohn disease, and can reduce the pain and side effects of patients in the treatment process while improving the curative effect. The invention provides technical support for preparing the medicine for treating the complicated anal fistula repair of Crohn's disease.
Description
Technical Field
The invention belongs to the technical field of biological medicines, relates to a human umbilical cord mesenchymal stem cell preparation, and in particular relates to a CD123 positive human umbilical cord mesenchymal stem cell preparation, a preparation method thereof and application thereof in treating Crohn's disease complicated anal fistula.
Background
Crohn's Disease (CD), also known as Crohn's disease, regional enteritis, crohn's enteritis, and granulomatous enteritis, is an inflammatory disease of the intestinal tract of unknown cause. Both this disease and chronic non-specific ulcerative colitis are collectively referred to as Inflammatory Bowel Disease (IBD). The etiology of Crohn's disease is unknown and may be a combination of pathogenic factors, related more closely to the avoidance of abnormalities, infections and genetic factors. Crohn's disease is a proliferative lesion through various layers of the intestinal wall and afflicts the mesentery and regional lymph nodes. Lesions are limited to 30% of the small intestine (mainly the terminal ileum) and colon, and they involve 40% of the colon lesions. Crohn divides pathological changes into acute inflammatory phase, ulcer formation phase, stenotic phase and phlegm tube formation phase (perforation phase). The lesions of this disease are distributed in segments, spaced apart from the normal intestine, and are well-defined and characterized by skip areas (skip areas). The clinical manifestations of the disease are quite various and are related to the location, scope, severity, duration and complications of the intestinal lesions. Typical cases often develop slowly in the young, with the course often ranging from months to years. The active period and the remission period are different in length and alternate with each other, and progressive progress is achieved in repeated attacks. A few acute diseases can be manifested by high fever, toxic blood symptoms and acute abdomen, and serious complications are more frequent. Sometimes, the symptoms are the first appearance of the intestines such as perianal abscess, phlegm tube formation, arthralgia and the like.
The chronic recurrent disease is a disease with unknown etiology, so that no effective therapeutic drug is developed, and currently, the drugs used for treating the Crohn disease mainly comprise biological agents such as glucocorticoids, salicylic acid preparations, immunosuppressants, antibiotics and the like, and the existing drugs can change symptoms of the disease to a certain extent, but can not completely relieve the disease condition and reduce the occurrence rate of complications, and the existing hormone drugs can cause damage to organisms and have obvious adverse reactions after long-term administration.
In regenerative medicine and clinical treatment, mesenchymal stem cells (MSC, mesenchymal stem cells) have wide clinical application prospects in various alternative treatments due to their ability to self-renew, differentiate in multiple lines and low immunogenicity. At present, no specific CD radical treatment method exists clinically, and along with the progress of the disease activity of patients, the effect of drug treatment is weakened, and the patients are easy to have adverse reactions such as allergy, drug resistance and the like. Thus, there is a need for a new method to replace traditional therapies that is safe and effective and that protects anal sphincter function. Finding new CD treatment regimens is of great clinical importance. In view of the limitations of the currently available treatments for Crohn's disease, it is imperative to find a new, safe, and fundamentally-safe method for treating Crohn's disease.
Disclosure of Invention
In view of the problems of the prior art directed to methods of treating crohn's disease, it is an object of the present invention to provide a human umbilical mesenchymal stem cell preparation that can be used to treat crohn's disease.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
A human umbilical cord mesenchymal stem cell preparation derived from fetal umbilical cord wharton's mesenchymal stem cells, said cell preparation being a CD123 positive cell preparation.
Further, the cell preparation positive for CD123 means that the cell flow detection cell preparation has a positive rate of CD123 of 90% or more, preferably 95% or more.
Further, the cell preparation has a positive rate of CD73, CD90 and CD105 of 90% or more, preferably 95% or more, as determined by cell flow assay.
Further, the cell preparation has a positive rate of CD14 and CD34 as determined by cell flow detection of less than 5%, preferably less than 2%.
Further, the preparation method of the cell preparation specifically comprises the following steps.
Step 1, culturing the Volton interval mesenchymal stem cells from the umbilical cord of the fetus by utilizing a serum culture medium to obtain the human umbilical mesenchymal stem cells.
And 2, incubating the human umbilical mesenchymal stem cells obtained in the step 1 with a CD123 antibody, and then sorting the obtained CD 123-positive cell preparation by flow cytometry.
Further, the serum concentration in the serum medium is less than 15vol%, the serum medium comprising a basal medium and a combination of growth factors and other substances that facilitate cell growth.
Still further, the serum concentration in the serum medium is 8 vol%.
Further, the composition of growth factors and other substances that facilitate cell growth includes: recombinant human granulocyte macrophage stimulating factor, recombinant human platelet-derived growth factor, recombinant human insulin-like growth factor, beta-nicotinamide mononucleotide, lycium barbarum polysaccharide, and fibronectin.
Further, the basal medium is DMEM high sugar medium.
Use of a CD123 positive human umbilical cord mesenchymal stem cell preparation in the preparation of a medicament for treating crohn's disease.
Further, the medicament is in the form of injection.
Further, the cell preparation has a CD126 positive rate of 90% or more, preferably 95% or more, as measured by cell flow assay.
Further, the cell preparation has a positive rate of CD73, CD90 and CD105 of 90% or more, preferably 95% or more, as determined by cell flow assay.
Further, the cell preparation has a positive rate of CD14 and CD34 as determined by cell flow detection of less than 5%, preferably less than 2%.
Further, the preparation method of the cell preparation specifically comprises the following steps.
Step 1, culturing the Volton interval mesenchymal stem cells from the umbilical cord of the fetus by utilizing a serum culture medium to obtain the human umbilical mesenchymal stem cells.
And 2, incubating the human umbilical mesenchymal stem cells obtained in the step 1 with a CD123 antibody, and then sorting the obtained CD 123-positive cell preparation by flow cytometry.
Further, the serum concentration in the serum medium is less than 15vol%, the serum medium comprising a basal medium and a combination of growth factors and other substances that facilitate cell growth.
Still further, the serum concentration in the serum medium is 8 vol%.
The specific process of subculturing primary humanized mesenchymal stem cells comprises the following steps: the obtained primary human umbilical cord mesenchymal stem cells are passaged into a culture dish for culture, and the human umbilical cord mesenchymal stem cells obtained by amplification in the culture dish after culture are first-generation human umbilical cord mesenchymal stem cells, namely P1 generation; and carrying out secondary subculture on the obtained P1 generation human umbilical cord mesenchymal stem cells to obtain the P2 and P3 generation human umbilical cord mesenchymal stem cells.
It will be appreciated by those skilled in the art that the actual dosage to be administered herein may vary greatly depending upon a variety of factors, such as the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.
Compared with the prior art, the invention has the following beneficial effects.
The CD123 positive human umbilical cord mesenchymal stem cell preparation provided by the invention does not influence the phenotype and differentiation capacity of MSC. The invention can fundamentally improve the inflammation of patients with the complicated anal fistula of the Crohn disease, and reduce the pain and side effects of the patients in the treatment process while improving the curative effect. The invention provides technical support for preparing the medicine for treating the complicated anal fistula repair of Crohn's disease.
Drawings
FIG. 1 is a morphological feature of cultured primary umbilical cord mesenchymal stem cells (. Times.100).
FIG. 2 is a morphological feature of umbilical cord mesenchymal stem cells (x 100) after passage expansion to passage 2 (P2).
FIG. 3 is a graph showing the detection result of the flow cytometer for detecting the markers related to the mesenchymal stem cells of the human umbilical cord.
Detailed Description
In order to facilitate understanding of the technical scheme of the invention, the following is a description of a CD123 positive human umbilical cord mesenchymal stem cell preparation, a preparation method thereof and application thereof in treating the complicated anal fistula of Crohn's disease with reference to the accompanying drawings and specific examples. The source of mesenchymal stem cells used in the present invention will be described: the various human tissues used in the invention are clinical wastes or isolated human tissues. For example, the umbilical cord in the examples is obtained from a female infant hospital obstetric ward in Shenyang city. The technical scheme of the invention does not relate to the specific operation of the process of acquiring human tissues. Unless otherwise indicated, all biochemical reagents used in the examples were commercially available, and the technical means used in the examples were conventional means well known to those skilled in the art.
The invention relates to a stem cell preparation, which is a CD123 positive human umbilical cord mesenchymal stem cell, wherein the cells express markers of the mesenchymal stem cell, such as CD73, CD90, CD105 and the like, and the positive rate is more than 90 percent; furthermore, this population of cells underexpresses leukocytes, endothelial cells, hematopoietic stem cell progenitors and, more importantly, CD123 positive cells hardly express markers of graft rejection, such as HLA-DR, indicating a very low likelihood of developing allergic reactions if such cell preparations are injected in the future.
In the present invention, CD 123-positive and CD 123-negative (hereinafter also referred to as CD 123) are isolated from fetal umbilical cord Wobbe-interval mesenchymal stem cells using CD123 as a marker - ) Two populations of cells were analyzed for markers on their surfaces by flow cytometry to find CD123 + And CD123 - The proportions of mesenchymal stem cell markers, graft rejection markers, leukocytes, endothelial cells, hematopoietic stem cell progenitors, and the like are nearly identical for both populations of cells except for CD 123.
The mesenchymal stem cells are marked by a plurality of markers, and each time the mesenchymal stem cells are identified, the mesenchymal stem cells need to be incubated with a plurality of antibodies, and a plurality of indexes are stained for final determination. The inventor of the present invention intends to find whether a new marker exists or not, and can replace other markers to become a new index for identifying mesenchymal stem cells, and the inventor of the present invention discovers the index of CD 123. CD123 is a subunit of the heterodimeric interleukin-3 receptor (IL-3R), which is a member of the beta co-receptor family. This family of membrane receptors plays an important role in regulating the growth, proliferation, survival and differentiation of hematopoietic cells, as well as immune and inflammatory responses. Prior to the present invention, it was generally believed that CD 123-negative cells would have a higher therapeutic effect on inflammatory enteritis than CD 123-positive cells, because CD 123-negative cells would not be stimulated by inflammatory factor IL-3, and the symptoms of inflammatory enteritis should be reduced. However, the inventors of the present invention have unexpectedly found the opposite conclusion that the therapeutic effect of the cell preparation that was positive for CD123 was surprisingly found to be far superior to that of cells that were negative for CD 123. The inventors of the present invention speculate that mesenchymal stem cells can secrete factors that inhibit inflammation under the stimulation of trace inflammatory factors, and have an anti-inflammatory effect against inflammation. The receptor of the CD123 positive cell surface expressed IL-3 can sense the stimulation of a trace amount of IL-3, immediately play an anti-inflammatory role and play a role in reducing inflammation; the cell surface negative to CD123 does not express IL-3 receptor, so that the stimulation of trace IL-3 is not felt, and the effect of resisting inflammation is not achieved. Thus, the therapeutic effect of CD123 positive cells is better.
The present invention relates to a cell preparation derived from fetal umbilical cord wharton's mesenchymal stem cells, which has a positive rate of CD73, CD90 and CD105 of 90% or more, more preferably 95% or more, as measured by cell flow assay.
The present invention relates to a cell preparation having a positive rate of CD14 and CD34 of less than 5%, preferably less than 2%, as determined by cell flow detection.
Wherein, the umbilical cord is developed from mesoderm, is a cord-like structure connecting fetus and mother in fetal period, is coated by amniotic membrane, and comprises two arteries and a vein and gelatinous tissue around blood vessel rich in proteoglycan and mucopolysaccharide. The Wobbe zone is gelatinous and is composed of fibroblasts, collagen fibers and proteoglycans. The fetal umbilical cord wharton's compartment mesenchymal stem cells are mesenchymal stem cells isolated from wharton's regions of the umbilical cord.
In the present invention, CD73, CD90 and CD105 are all surface markers commonly used for identifying mesenchymal stem cells, and the positive rate of CD73, CD90 and CD105 proves that the cell preparation is mesenchymal stem cells and has high purity.
In the present invention, the markers of CD14 and CD34, CD14 and CD45, CD34 and CD34 hematopoietic stem cells, respectively, are used as markers of endothelial progenitor cells, and the low positive rate of the three markers of CD14 and CD34 indicates that the cell preparation of the present invention has high purity.
The purity of the umbilical cord Wobbe interval mesenchymal stem cell preparation prepared by the invention is equivalent to that of umbilical cord mesenchymal stem cells cultured by 10vol% FBS, and the low serum culture medium is proved to be practical for culturing human umbilical cord Wobbe interval mesenchymal stem cells.
In the present invention, the above cell preparation is detected by flow cytometry, specifically, umbilical cord Walton interval mesenchymal stem cells are digested with pancreatin and counted, 5×10 cells per cell 5 The total amount was 11 parts. One of them was used as a negative control and three were used as Shan Yangguan for three fluorescent dyes, FITC, PE and APC, respectively (4 samples above were used to debug flow cytometry, i.e. to adjust voltage and compensate). And 7 parts of cells are taken as sample tubes, antibodies of CD73, CD90, CD105, CD14, CD34, CD123 and HLA-DR marked by one of three fluorescent dyes of FITC, PE or APC are respectively added, the cells are incubated on ice for 20 minutes, excessive antibodies are washed off, 200 microliters of PBS buffer solution is added to each tube to resuspend the cells, and the positive rate of each index of the antibodies of CD73, CD90, CD105, CD14, CD34, CD123 and HLA-DR can be obtained by analyzing the samples through a flow cytometer.
In the present invention, the flow cytometer to be used is not particularly limited, and any flow cytometer commonly used in the art may be used.
The cell preparation of the present invention is prepared by a method comprising the steps of: umbilical cord wharton's space mesenchymal stem cells from fetal umbilical cord are cultured with a low serum medium to obtain an umbilical cord wharton's space mesenchymal stem cell preparation.
The method further comprises obtaining wharton's interval mesenchymal stem cells from the umbilical cord. The method for obtaining the wharton's jelly stem cells from the umbilical cord is not particularly limited, and any method conventional in the art may be used. In a specific embodiment, primary umbilical cord mesenchymal cells are obtained using a tissue mass culture method. I.e. the umbilical cord Wobbe zone tissue is reduced to 1cm by using ophthalmic scissors 3 Small pieces of tissue were spread in 10cm dishes with forceps and low serum as described herein was addedThe culture medium was cultured in an incubator at 37℃for about 2 weeks, and the cells were allowed to climb out.
In the present invention, a low serum medium is used for the culture. In general, in the context of mesenchymal stem cell culture, serum concentrations are different from one researcher or institution to another. Serum contains various plasma proteins, polypeptides, carbohydrates, growth factors, hormones, etc. Serum components are complex, and each batch of serum has differences, so that the consistency of the components cannot be ensured. In addition, although serum contains many components that are favorable for cell growth, it is inevitable to contain some components that are harmful to cells, such as complement, antibodies, endotoxins, etc. Thus, cells cultured with high concentrations of serum are not suitable for clinical use and increase the risk of clinical allergy. The low serum culture medium has no adverse effect on cell growth, proliferation, morphology and function. The low serum medium of the present invention includes basal medium, growth factors and compositions of matter that promote cell growth.
The above volume percentages (taking medium containing 8% FBS as an example) refer to the addition of 8 ml of Fetal Bovine Serum (FBS) and 92 ml of basal medium, i.e. the ratio of FBS to total medium volume, per 100 ml of medium prepared.
The basal medium refers to a medium comprising carbohydrates, amino acids, vitamins and inorganic salts.
In a preferred embodiment of the present invention, DMEM high sugar medium is used, which is capable of providing nutrients necessary for the survival of mesenchymal stem cells in vitro, including four major classes of substances, namely carbohydrates, amino acids, vitamins and inorganic salts. DMEM medium is developed on the basis of MEM medium, and has higher nutrient content than MEM and alpha-MEM.
The composition of the above growth factors and various substances that facilitate cell growth includes: recombinant human granulocyte macrophage stimulating factor, recombinant human platelet-derived growth factor, recombinant human insulin-like growth factor, beta-nicotinamide mononucleotide, lycium barbarum polysaccharide, and fibronectin.
The low serum medium used in the present invention comprises: recombinant human granulocyte macrophage stimulating factor 1-150 ng/ml (preferably 1-20 ng/ml), recombinant human platelet-derived growth factor 1-150 ng/ml (preferably 1-20 ng/ml), recombinant human insulin-like growth factor 1-40 ng/ml (preferably 1-20 ng/ml), beta-nicotinamide mononucleotide 1-100 μg/ml (preferably 1-20 μg/ml), lycium barbarum polysaccharide 1-100 μg/ml (preferably 1-30 μg/ml), fibronectin 1-100 ng/ml (preferably 5-20 ng/ml), FBS 1-15vol% (preferably 8-15 vol%), 0.3-0.7 vol% of an aqueous solution of non-essential amino acids (preferably 0.5 vol%). In addition, the DMEM high-sugar medium used in the present invention was supplemented with 2mmol/L of L-glutamine, 50U/ml penicillin, 50. Mu.g/ml streptomycin.
The stem cell preparation of the present invention is a preparation obtained by culturing umbilical cord Wobbe cells of a fetal umbilical cord in a low serum medium. The cell preparation of the invention is detected by a flow cytometry, and the cell preparation of the invention is found to highly express markers of stem cells such as CD73, CD90, CD105 and the like, and the proportion of the stem cell markers is more than 90 percent; in addition, the cell preparation of the invention expresses hematopoietic and endothelial cell markers at low levels, and the ratio of CD14 to CD34 is less than 5%; more importantly, the cell preparation does not express the HLA-DR marker related to the transplant rejection, the cell preparation has higher purity and extremely low risk of the transplant rejection.
Furthermore, the method of the invention for producing a CD123 positive cell preparation further comprises: the umbilical cord wharton's cell preparation obtained as described above was incubated with CD123 antibody and then CD 123-positive cell preparation obtained was sorted by flow cytometry. After incubation, aseptically sorting CD123 positive cells and CD123 negative cells by flow cytometry with CD123 as sorting markers to obtain CD123 positive umbilical cord wharton's cell preparations with a CD123 ratio of more than 90%, preferably more than 95%; a CD 123-negative umbilical cord Wangton's interval mesenchymal stem cell preparation is obtained, wherein the proportion of CD123 is below 10%, preferably below 5%.
The CD123 positive human Wobbe umbilical cord mesenchymal stem cell preparation can effectively inhibit the weight reduction of the mouse with the complicated anal fistula due to the Crohn's disease, effectively inhibit the disease activity index of the mouse with the complicated anal fistula due to the Crohn's disease and effectively prolong the colon length of the mouse with the complicated anal fistula due to the Crohn's disease. It can be seen that the cell preparation of the present invention is effective in treating inflammatory Crohn's disease complicated with anal fistula in mice.
In DSS-induced complicated anal fistula with rohn's disease, CD123 is infused intravenously + After the cell preparation, the hematochezia, the loose stool and the weight reduction degree of mice induced by the DSS can be obviously reduced, in addition, the colon length of the mice in a cell treatment group is obviously higher than that of the mice in a DSS treatment group, the mice can effectively treat the anofistula complicated with the Roen disease, and the CD123 is screened under the same condition - Cells have no obvious treatment effect on the anal fistula complicated with the Roen disease. Therefore, when the cell preparation is used for treating inflammatory enteritis, the marker is used as a detection standard, and other markers are not needed to be detected, so that time and labor are saved.
The cell preparation of the present invention is prepared using a low serum medium, and culturing using the low serum medium has no adverse effect on cell growth, proliferation, morphology and function.
The cell preparation of the invention can be used for treating inflammatory bowel disease, for example, ulcerative colitis and Roen's disease complicated anal fistula.
The present invention relates to the use of the above-described cell preparation for the manufacture of a medicament for the treatment of inflammatory bowel disease, and inflammatory bowel disease-related complications and diseases of similar pathogenesis, including but not limited to irritable bowel syndrome, arthritis and other extra-intestinal complications including ankylosing spondylitis, pyoderma gangrenosum, erythema nodosum, iritis, uveitis, episcleritis and primary sclerosing cholangitis.
Example 1 preparation of human umbilical cord mesenchymal stem cell preparation.
The composition of the low serum medium used in the examples is as follows: recombinant human granulocyte macrophage stimulating factor 15ng/ml, recombinant human platelet-derived growth factor 10ng/ml, recombinant human insulin-like growth factor 20ng/ml, beta-nicotinamide mononucleotide 15 μg/ml, lycium barbarum polysaccharide 30 μg/ml, fibronectin 15ng/ml,0.5 vol% non-essential amino acid aqueous solution, 2mmol/L L-L-DMEM high-sugar medium supplemented with 8-15% FBS,50U/ml penicillin, 50 μg/ml streptomycin.
The human umbilical cord mesenchymal stem cells are isolated and cultured as follows.
(1) The small pieces of umbilical cord tissue were sheared off and washed in Phosphate Buffered Saline (PBS) in sterile cups until clear, at which point the wash should be transparent.
(2) Umbilical cord tissue was soaked with 75% alcohol for 3 minutes and washed twice with PBS containing 10% diabody to remove alcohol residues. 2-3cm umbilical cord tissue was cut by scissors and placed in 10cm sterile petri dishes.
(3) Removing vein wall, two arteries and amniotic membrane with hemostatic forceps, and stripping Whatman's jelly. Stripping about 2g of Whatman's jelly, transferring into a 50ml centrifuge tube, and shearing.
(4) 800g, centrifuging for 5min, discarding supernatant, and uniformly plating the tissue blocks.
(5) After 24 hours, 10ml of complete medium of 8% FBS was added and the culture was continued in a 5% CO2 incubator at 37 ℃.
(6) The primary cells grew for about 15-20 days, and as shown in fig. 1 when observed under a microscope, the spindle-shaped adherent cells were found to grow around the tissue mass, and passaging was possible.
(7) The culture broth was aspirated, washed twice with PBS to remove the remaining medium, 3ml of pancreatin was added, and after the cells were in suspension, 5ml of stop solution was added.
(8) The cell suspension was transferred to a 50ml centrifuge tube and the flask was rinsed with saline. Centrifugation at 1000rpm for 5min, the supernatant was discarded and the wash was repeated once.
(9) Resuspended cells were blown with complete medium, and 100 micron mesh was filtered to single cell suspension while the tissue mass was removed. 1 st pass 2 or 1 st pass continued to culture in a 37℃5% CO2 incubator.
(10) Fresh DMEM medium containing 8% fetal bovine serum was then changed every 3 days and the cells were observed under a microscope as shown in fig. 2.
In the invention, the operations of extracting, culturing and establishing the cell bank of the umbilical mesenchymal stem cells are all completed in a GMP production workshop of the company. The cells are subjected to separation culture and passage amplification to 2 nd generation (P2) and then are subjected to detection of exogenous microorganisms, viruses, endotoxin and the like; and simultaneously detecting the immunophenotype, the differentiation capacity, the biological efficacy of the cells and the like. And (5) taking the qualified cells as seed bank cells, and placing the seed bank cells in a liquid nitrogen tank at the temperature of-196 ℃ for preservation.
The umbilical cord mesenchymal stem cells are obtained by adopting the culture method. Specifically, human umbilical cord mesenchymal stem cells isolated by a tissue mass method are cultured by the serum culture medium, when about 80% of the cells are combined, 0.25% of the cells are digested for 1-2 minutes, most of the cells are used for expansion culture, and a small part of the cells are used for identifying markers of the stem cells. The expression of CD73, CD90, CD105, CD14, CD34, CD123 and HLA-DR was examined by flow cytometry (after cells were incubated with the above corresponding antibodies, excess antibodies were washed off, the cells were resuspended with PBS, and the positive rates of the above markers were examined, as shown in FIG. 3. The results of the experiment in FIG. 3 show that the obtained cell preparation highly expressed markers of mesenchymal stem cells, CD73, CD90 and CD105 were all over 90%, the ratio of low expressing hematopoietic stem cells and endothelial cell markers CD14, CD34 was less than 5%, and the cell preparation did not express HLA-DR, a marker of pre-transplant rejection.
The obtained umbilical cord Walton interval mesenchymal stem cell preparation is incubated with CD123 antibody for 20min at 4 ℃, excess antibody is washed off, and cells are resuspended with PBS. Using CD123 as a sorting marker, and aseptically sorting CD123 positive cells and CD123 negative cells by a flow cytometry to obtain a CD123 positive umbilical cord Wobbe interval mesenchymal stem cell preparation, wherein the proportion of the CD123 is 99.1%; a CD 123-negative umbilical cord Wangton interval mesenchymal stem cell preparation was obtained, the proportion of CD123 was 0.8%.
Example 2 treatment of mouse DSS-induced roen disease with CD123 positive mesenchymal stem cells anal fistula was complicated.
C57BL/6 mice were acclimatized for 7 days and were randomized into four groups, namely a normal control group, a DSS model group (DSS, sodium dextran sulfate, a modeling agent for enteritis model), a CD123 positive cell preparation treatment group and a CD126 negative cell treatment group. Mice were given 3.5% DSS water for 7 days, cellsTreatment groups were injected 1x10 by tail vein on 1,3,5 days of DSS water, respectively 6 The injection volume of each CD123 positive cell preparation or CD123 negative cell was 100 μl/unit. DSS model group, 100 μl/PBS buffer was injected from the tail vein on 1,3,5 days of DSS water, respectively. Mice were weighed daily and tested for hematochezia. At the end of the experiment, mice were sacrificed, the whole colon was taken, the length thereof was detected, and the body mass index and the colon length of the mice were detected, the colon length of the mice treated with the CD 123-positive cell preparation was significantly higher than that of the mice treated with the model group and the CD 123-negative cell preparation, which was close to the result of the normal control group, showing that the CD 123-positive cell preparation obtained by the present invention has the effect of treating enteritis; statistical analysis of colon length in each group of mice also showed that the CD123 positive cell preparation obtained according to the invention had the effect of treating Luo Enbing concurrent anal fistula.
After the experiment is finished, the mice are anesthetized, blood is taken for separating serum, and the level of inflammatory factors in the serum is detected by an enzyme-linked immunosorbent assay (ELISA method). After DSS induction, the levels of serum inflammatory factors IL-3 and TNF-alpha of mice are obviously increased, and CD123 positive cell preparations are given, so that the levels of inflammatory factors IL-3 and TNF-alpha can be obviously reduced, and CD123 negative cells have no obvious inhibition effect on inflammatory factors, so that the CD123 positive cell preparations have the effects of reducing inflammatory factors and inhibiting anal fistula complicated with Ron disease.
The abdominal cavity of the above mouse is opened, the whole colon of the mouse is taken, the feces is washed off, and colon tissue is subjected to formalin fixation, gradient alcohol dehydration, xylene transparency, paraffin embedding, lycra slicing (thickness of 4 μm), dewaxing, rehydration, hematoxylin-eosin staining, dehydration, transparency and neutral resin sealing. The colon tissue of the normal mouse has orderly cell arrangement, clear tissue structure and no inflammatory cell infiltration. DSS-induced model group mice have colonic tissue, disordered cell arrangement, disordered tissue structure and massive inflammatory cell infiltration. After injection of the CD123 positive cell preparation, the tissue structure of the colon was essentially normal with a small amount of inflammatory cell infiltration. After injection of CD123 negative cells, the tissue structure of the colon was still disordered, but slightly lighter than the model group, with massive inflammatory cell infiltration. Therefore, the CD123 positive cell preparation obtained by the invention can effectively inhibit colon structural disturbance caused by DSS, inhibit inflammatory cell infiltration, and further effectively treat the complicated anal fistula of Roen disease.
On the basis of the treatment of the experimental animals, 5 patients with Crohn's disease and 5 patients with ulcerative colitis are treated, and similar symptoms appear before treatment for the patients with Crohn's disease with ages of 27 years, 34 years, 44 years, 52 years and 60 years, and diarrhea with blood, weight loss and abdominal cramp appear 2-5 times a day.
The present invention is not limited to the preferred embodiments, and those skilled in the art will appreciate that many modifications, adaptations and variations of the present invention are possible in light of the above teachings without departing from the scope of the present invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
Claims (12)
1. A human umbilical cord mesenchymal stem cell preparation, wherein the cell preparation is derived from fetal umbilical cord wharton's jelly stem cells, and the cell preparation is a CD123 positive cell preparation.
2. The human umbilical mesenchymal stem cell preparation of claim 1, wherein the cell preparation positive for CD123 is a cell preparation with a positive rate of CD123 of 90% or more detected by cell flow.
3. The human umbilical mesenchymal stem cell preparation of claim 1, wherein the cell preparation has a positive rate of CD73, CD90 and CD105 of 90% or more as determined by cell flow detection.
4. The human umbilical mesenchymal stem cell preparation of claim 1, wherein the cell preparation has a positive rate of CD14 and CD34 of less than 5% as determined by cell flow assay.
5. The method for preparing the human umbilical cord mesenchymal stem cell preparation as claimed in any one of claims 1 to 4, which specifically comprises the following steps:
step 1, culturing the Volton interval mesenchymal stem cells from the umbilical cord of a fetus by utilizing a serum culture medium to obtain human umbilical mesenchymal stem cells;
and 2, incubating the human umbilical mesenchymal stem cells obtained in the step 1 with a CD123 antibody, and then sorting the obtained CD 123-positive cell preparation by flow cytometry.
6. The method of claim 5, wherein the serum concentration in the serum medium is less than 15 vol.% and the serum medium comprises a basal medium and a combination of growth factors and other substances that facilitate cell growth.
7. The method according to claim 6, wherein the serum concentration in the serum medium is 8 vol%.
8. The method of claim 5, wherein the composition of growth factors and other substances that facilitate cell growth comprises: recombinant human granulocyte macrophage stimulating factor, recombinant human platelet-derived growth factor, recombinant human insulin-like growth factor, beta-nicotinamide mononucleotide, lycium barbarum polysaccharide, and fibronectin.
9. The method of claim 5, wherein the basal medium is DMEM high sugar medium.
10. Use of CD123 positive human umbilical cord mesenchymal stem cell preparation in the preparation of medicament for treating crohn's disease complicated anal fistula.
11. The use according to claim 10, wherein the medicament is in the form of an injection.
12. The use of claim 10, wherein the cell preparation has a CD126 positive rate of 90% or more as measured by cell flow assay.
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