Expirical Study Of Bone Marrow Mesenchymal Stem Cellular Transplant For The Repair Of Ulcerative Colitis

Objective: Bone marrow mesenchymal stem cells(BMSCs) are considered to maintain the high proliferative capacity, have the potential to differentiate into all cells in three germinal layers, and is useful in the fields of tissue engineering, wound repair and cell transplant therapy. At present, it had been reported that transplantation of BMSCs can be the new effective therapy for inflammatory bowel disease, nevertheless, the theory about distribution of transplanted BMSCs and treatment mechanism in colon is not clear. This study try to compare several UC models to find the most proper one; to isolate, culture, amplificate, mark the rat BMSCs with fluorescence, and observe the bionomics of BMSCs; to observe the distribution of the marked BMSCs in different organs and tissues at different time points after transplantation of BMSCs in UC model of rats. Whether or not the number of BMSCs in colon is more than in other segments of digestive canal? the number of BMSCs in ulcerative focus of infection of colon is more than in non-ulcerative places? Whether or not the repair of injured tissue in UC models after transplanting is better than without transplanting? Whether or not EGF can encourage BMSCs moving to ulcerative focus of infection? Try to provide the basic theory and experiment for the future research of BMSCs in the tissue engineering and treating intestinal mucosa injury disease.PARTⅠTHE ESTABLISHMENT AND COMPARING OF ULCERATIVE COLITIS MODEL IN RATS1. Materials and Methods1.1 SD rats were randomly distributed into groupⅠ(immune- combined TNBS/ethanol), groupⅡ(TNBS/ethanol), groupⅢ(50% ethanol) and groupⅣ(normal saline). The model were made according to literatures, rats were sacrificed at day 1, 21 and week 8, 12 after enema with different material in 4 groups to observe the macroscopical focus of infection in bowels, the microscopical changes and the expressing-level of TNF-αand IL-10 in mucous membrane of colon.2. Results2.1 The observation with naked eye and light microscope show that the typical erosion and ulcer changes appeared in colon of groupⅠandⅡ, and there was accompanying pathological changes in the end piece of ileum in groupⅠ. The pathological changes for groupⅠcould keep for 8 weeks, while those of groupⅡtrended to heal 3 weeks later. The pathological changes in groupⅢonly were hyperemia, oedema and a few anabrosis or superficial ulcer, keeped for 21days. There was no tissue damage in colon of groupⅣ. 2.2 Immunohistochemistry revealed: the level of TNF-αin mucous membrane of colon of groupⅠ,Ⅱ,Ⅲwere much higher than that of groupⅣ, but the expression of IL-10 were lower. The difference of expression of TNF-αand IL-10 was statistical significance between groupⅠ,Ⅱ,Ⅲand groupⅣ(P<0.01).PARTⅡTHE BIOLOGIC CHARACTER AND LABELLING OF BMSC1. Materials and Methods1.1 Obtain limbs bone marrow samples from SD rats aged 4 to 6 weeks. BMSCs had been isolated, cultured and proliferated in vitro. The adherence rate, growth curve of BMSCs had been observed. The express of CD44, CD45, CD90 and status of cell cycle in the 3th and 5th generation of BMSCs ( P3 and P5 ) are detected by flow cytometry.1.2 After Hoechst33342 (HCT)f luorescent labelling P3-BMSCs, the marking condition in different times were observed by fluorescence microscope, and the marking rates were calculated. The growth information of BMSCs after freezed and resusing in common practice has been observed.2. Results2.1 The primary cells can adherence easily,the average rate of living cell is 95.6±1.4%. The BMSCs proliferated rapidly from cultured 3 to 12 days, have typical feature of spindle-shape and whirlpool arrangement in culture. The subcultured cells proliferated faster than primary cells. The detention period of BMSCs is 24~48h, the log period is 3~6d, the platform period is 7~8d. The BMSCs can proliferate more than 2.2 times in DMEM-F12 (contain 10%FBS). The primary revivaled BMSCs grew slowly, and after subcultured, the cells restored growth condition as before freezing. After 6 generation, BMSCs appear the signs of senility.2.2 The result of flow cytometer–detection showed that the BMSCs in generation 3 and 5 express CD44, CD90, but not express CD45. Cell cycle status analysis revealed that a small population of the cells were actively engaged in proliferation (S+G2+M=9.12%), and more than 90.81% of cells were in G0/G1 phase.2.3 The BMSCs marked with HCT proliferated normaly, the marking rate of the cells decreased slowly, which could be about 90% after the fourth week.PARTⅢTRANSPLANT AND REPAIR EFFECT OF BMSCS IN RAT ULCERATIVE COLITIS MODEL1. Materials and Methods1.1 SD rats were randomly distributed into 4 groups, Colitis was induced in group A, B and C with immune- combined TNBS/ethanol. Following the induction of colitis, each rat in 4 groups received caudal vein injection of 1 ml different fluids, which were: normal sodium for group A, HCT marked BMSCs(1×106)for group B and D, HCT marked BMSCs (1×106)with 300μg for group C.1.2 5 Rats were sacrificed at day 3, 7 and 14 after injection in each group, dissected and observed the colonic pathological changes. The bone marrow, lung, spleen, liver, esophagus, stomach, small intestine and colon of group B, C and D were prepared as frozen sections to observe the distribution of HCT marked BMSCs in these organs with fluorescent microscope. The value of integrated optical density (IOD) in each group and different time points were detect in 10 fields of vision of 20 times object lens,to survey the quantitive discrepancy of HCT marked BMSCs in these organs.1.3 The colon in groups A, B, C and D were prepared as paraffin section stained by HE to observe the microscopical pathological-changes. Another parts paraffin sections were stained by immuno histochemistry method to observe the express of TNF-αand IL-10 in colon.2. Results2.1 There was a regular time pattern about IOD level of BMSCs in bone marrow, lung, spleen and liver: IOD level of BMSCs in lung, spleen and live was 7d > 3d > 14d;the IOD level of BMSCs in bone marrow was 7d > 14d > 3d. At day 7, the highest IOD of BMSCs was in lung in all organs, and at day 14, the highest one was in bone marrow(P<0.05). The difference of the IOD level in same organ at same time point in all groups did not showed the statistically significant.2.2 On the 3, 7 and 14d, HCT marked BMSCs in groups B, C and D were seen in mucosa layer of esophagus, stomach, ileum. There was no statistically significant among the difference in the IOD level of esophagus, stomach, ileum at same time point in all groups.2.3 On the 3 day of transplantation, the difference of the IOD level between colon and esophagus, stomach, ileum in same groups did not showed the statistically significant. But, on the 7, 14 day of transplantation, the IOD level in group B and C were higher than that in group D(P<0.05). The difference of the IOD level between group B and C had no statistically significant.2.4 Under light microscope, the HCT marked BMSCs were seen mainly in colonic mucosa of group B, C and D in each time point, occasionally were seen in submucosa and tunica muscularis. On the 14 day of transplantation, the marked BMSCs in diseased region of colon was maximum, more number of marked BMSCs was appeared in the mucosa anabrotic focus of infection, and those in the peripheric mucosa of ulcer was exceed than that in colonic mucosa of non-anabrotic focus of infection.2.5 There was macroscopic and microscopic evidence of colonic mucosa congestion, edema, epithelial necrosis, inflammatory cell infiltration and ulcer in group A, B and C, but no one be noted in group D. The extent of reparation of colonic mucosa-injury in group B and C were surpassed obviously than that in group A at day 14, but there was no difference between group B and C.2.6 At day 3, 7, 14 of transplantation, the expression of TNF-αin mucous membrane of colon in group A, B, C were higher than that in group D, but the expression of IL-10 lower than that in group A(P<0.01) . At day 14, the expression-level of TNF-αin colonic mucosa in group B, C were lower than that of group A, but the expression of IL-10 were opposite (P<0.01). The difference of expression of TNF-αand IL-10 was no statistically significant.Conclusion of whole article1. Immune-combind TNBS/ethanol method was the ideal method to establish the UC model comparatively.2. The rats BMSCs have been isolated successfully by adherence screening. The result of Flow cytometer analyzed had shown that the cells of passage 3, 5 possess the character of surface antigens of MSCs and the cell cycle feature of stem cells. The subcultured cells proliferated faster than primary cells; The BMSCs could proliferate more than 2.2 times in DMEM-F12 (contain 10% FBS). The grow of BMSCs was not effected by freezing.After 6 generation, BMSCs appear the signs of senility.3. HCT marked cells was a useful method for tracing BMSCs in short period.4. Transplanted BMSCs widely distributed throught body. BMSCs in lung at day3, 7 and in bone marrow at day 14 were higher than that in liver and spleen. It hinted that most BMSCs retained in blood circulation.5. Transplanted BMSCs could migrate and distribute in mucosa of colon on day 3. The number of BMSCs in injured colon was more than that in esophagus, stomach, ileum and nomal colon. The number of marked BMSCs in the mucosa of anabrotic focus of infection was more than that in normal colonic mucosa. It indicated that BMSCs may trended to migrant into injured tissue.6. The extent of reparation of colonic mucosa-injury in BMSCs transplanting group was surpassed obviously than that in non- BMSCs transplanting group. It show us that transplantation of BMSCs could repair damage or promote repairing damage in colon.7. The higer expression of IL-10 and the lower expression of TNF-αin colinic mucosa of BMSCs transplanted UC rats indicated that BMSCs migrated in colonic mucosa of UC rat might be regulate the inflammatory cell factors, TNF-αand IL-10, to achieve immunologic balance, thus,promote the recovery of colonic focus of infection in UC rat.8. Between BMSCs transplantation groups with EGF or without EGF, there was no difference in the number and the distribution of BMSCs in gut or injured colon and the expression of inflammation factors. It indicated that injection of EGF once could not encourage BMSCs migranting to the injured tissue or repairing the damage of colonic tissue.