Treating Effect Of Transplanted Bone Marrow-Derived Mesenchymal Stem Cells For Ulcerative Colitis Of Rats

Ulcerative colitis(UC) is a chronic relapsing disease of inflammatory bowel disease(IBD). The incidence and prevalence of UC was increased in recent years. Currently, the therapeutic approaches for UC could alleviate the symptoms of UC, but have obviously side effects and can't cure the disease. Therefore,it is urgent to develop some new therapeutic approaches that are more effective and less side effects for UC. Mesenchymal stem cells(MSCs) are considered to maintain the high self-renewal capacity, have the potential to differentiate into all cells of three germinal layers, and be useful in the fields of stem cell transplanting therapy and tissue engineering. At present, it had been reported that transplantation of MSCs could be a new effective therapy for IBD. Nevertheless, the differentiation of transplanted MSCs in intestinal tract and the treatment mechanism are not clear. This study try to isolate, culture, proliferate the MSCs of rats, and then transplant the cells labeled with DAPI into the UC rats model through their caudal vein, to observe the therapeutic effect and explore the possible mechanisms. PARTⅠThe isolation, culture and identification of rat bone marrow-derived mesenchymal stem cells1 Materials and Methods1.1 Bone marrow of rats aged 3 to 4 weeks was flushed out of tibias and femurs. MSCs were isolated, cultured and proliferated by adherence screening in vitro. The morphological characteristics and growth of primary culture cells and passage cells were observed under inverted phase contrast microscope. The expression of CD29, CD90 and CD45 in the 3th generation of MSCs ( P3-MSCs ) were detected by flow cytometry.1.2 The osteogenic differentiation and adipogenic differentiation were performed in P3-MSCs2 Results2.1 The primary MSCs began to be adhesive after culture for 3～4h. The cells proliferate rapidly after culture for 3～12d, had typical feature of spindle-shape and whirlpool arrangement. The passage cells proliferated faster than primary cells. The detention period was 24~48h, the log period was 3~6d and the platform period was 7~8d in the P3-MSCs. The MSCs appeared the signs of senility after 6 generations. The results of flow cytometer–detection showed that the P3-MSCs expressed CD29, CD90, but not expressed CD45. The homogenicity of CD29 and CD90 were 99.82±0.11 % and 99.84±0.25% respectively。 2.2 After osteogenic induction, most MSCs became polygon or irregular shape, and calcium depositions could be observed in their cytoplasma after Von Kossa staining; The cells in control group were still fusiform, had no calcium deposition appeared. The MSCs became round or oval after adipogenic induction, and lipid droplets were observed in their cytoplasma after oil Red O staining; The cells in control group maintaind their fusiform, had no lipid droplets appeared. PARTⅡTherapy of transplanted bone marrow-derived mesenchymal stem cells for ulcerative colitis of rats1 Materials and Methods1.1 The were labelled with DAPI.1.2 SD rats were randomly distributed into 3 groups: transplantation group, PBS group and normal control group. Colitis was induced by immune-combined TNBS/ethanol in transplantation group and PBS group. Following the induction of colitis, 1mL fluids was injected through caudal vein of rats: DAPI labeled P3-MSCs (3×106) in transplantation group;PBS in the other two groups.1.3 Symptoms and signs of rats were observed daily. 5 rats were sacrificed at day 3, 7 and 14 after injection in each group, and obtained the injured colon. And the colonic tissue were prepared as paraffin sections to observe the pathological changes under optical microscope. 1.4 The colonic tissue in transplantation group at different times were prepared as frozen sections to observe the distribution of transplanted cells under fluorescent microscope. The cell numbers of DAPI labelled MSCs were counted randomly in 10 fields of high magnification(×200). The expression of cytokeratin(CK) in transplanted MSCs were observed under laser confocal microscopy after immunofluorescent staining.2 Results2.1 Blue flourescence was observed in the nucleus of MSCs after labelling with DAPI. There were about 90% cells labelled with DAPI after 14 days.2.2 The clinical symptoms of rats in transplantation group was significantly ameliorated. The paraffin sections of colon in transplantation group shown that the reparation of injured colon was obviously surpassed than that in PBS group at day 7 and 14.2.3 The labelled cells could been seen in the injured colon in transplantation group at day 3, 7, 14, and mostly in the mucosa and submucosa, occasionally in the muscular layer and adventitia. There were more labelled cells in the peripheric mucosa of ulcer than those in normal colonic mucosa. The results of statistics revealed that the number of labelled MSCs in transplantation group was increased gradually,and was maximum on day 14(P<0.05). After 7 and 14 days of postgraft, some MSCs labelled with DAPI in colonic tissue could express CK. Conclusion of whole article1. The rats MSCs have been isolated, cultured and proliferated successfully by adherence screening. The results detected by flow cytometer had shown that the passage 3 cells expressed the phenotype characteristics of MSCs.2. P3-MSCs could be induced to differentiate into osteogenic cells and adipogenic cells. It demonstrated that the isolated MSCs had multipotent differentiation capability.3. There was obvious therapeutic effect of transplanted MSCs for ulcerative colitis of rats.4. The labelled MSCs could be seen in the colon of rats after 3,7,14 days of postgraft. There were more labelled cells in the peripheric mucosa of ulcer than those in normal colonic mucosa. These results indicated that the injured tissue and organ may have a specific chemotaxis for MSCs.5. Part of MSCs in colonic tissue could express CK, which indicated that the transplanted MSCs could diferentiate into epithelial cells to participate the repair of ulcerative colitis of rats.