The Distribution Of Intravenous Transplantated Bone Marrow Mesenchymal Stem Cells In Ulcerative Colitis Rat Model

Objective: Bone marrow mesenchymal stem cells(BMSCs) have the characteristic to maintain the high proliferative capacity, and the potential to differentiate into cells in three germinal layers, and is useful in the fields of tissue engineering, wound repair and cell transplanted therapy. Following the utilized research of BMSCs for tissue repair and gene therapy, the migration potential of targeted BMSCs in vivo or cultured in vitro has caused the interesting of scientists. In this study, BMSCs of rats will be isolated, cultured and labelled with fluorescent-DAPI, and then, transplanted into the ulcerative colitis (UC) rat model through caudal vein, to investigate the effects of transplanted cells concentration and sodium nitroprusside on the BMSCs homing to colon of UC rats. 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 biologic character of BMSCs1. The bone marrow was obtained samples from SD rats aged 4 to 6 weeks. BMSCs had been isolated, cultured and proliferated in vitro. The express of CD44, CD45, CD90 in the 3th and 5th generation of BMSCs ( P3 and P5 ) are detected by flow cytometry. Results: The BMSCs proliferated rapidly from cultured 3 to 12 days, have typical feature of spindle-shape and whirlpool arrangement in culture. The result of flow cytometer–detection showed that the BMSCs in generation 3 and 5 express CD44, CD90, but not express CD45. Their homogenicity of expression CD44, CD90 was 98.26±0.48% and 98.68±0.32% respectively.2. Adipogenic differentiation was induced by culturing P3-BMSCs in adipogenic medium ( including 10%FBS, 1μmol/L dexamethasome, 0.5 mmol/L of 3-isobutyl-1-methyl-xanthine, 10 mg/L insulin and 100mmol/L indomethncin in H-DMEM ) . On the 3rd day of inducting, it could be seen that some cells become round or oval, and lipid droplets could appeared in their cytoplasma. After inducted 10 day, an oil Red O staining showed that the lipid droplets in cytoplasma were stained as orange, but the cells in control group was still fusiform, no lipid droplets appeared.Osteogenic differentiation was performed through culturing P3-BMSCs in osteogenic medium (10% FBS, 0.1μmol/L dexamethasome, 10mmol/Lβ-glycerophosphate,50mg/L vitamin C in L-DMEM). From the 2nd day of inducting, some cells become cuboidal. On the 8th day of inducting, most cells become polygon form, and on the 19th day, the calcium deposition stained by Alizarin Red S appeared orange. But the cells in control group was still fusiform, no calcium deposition appeared.PARTⅡThe distribution of Transplanted-BMSCs in ulcerative colitis rat model1. SD rats were randomly distributed into A, B and C groups. The ulcerative colitis rat model was induced with immune-combined TNBS/ethanol in 3 groups. The suspension of fluorescent-DAPI labeled P3～P5-BMSCs were prepared for cells transplantation.Following the induction of colitis, each rat in 3 groups was received caudal vein injection of 1mL fluids contained 1×106,5×106,1×107 DAPI marked BMSCs respectively. After injection, 5 rats in each group were sacrificed at day 7 and 14, and their colon were prepared as cryostat section. The cell numbers of DAPI marked BMSCs were counted with fluorescent microscope in the colitis tissue and normal colonic tissue respectively.The same section was stained by HE to observe the microscopical changes of pathology.Results: On the 7th day and 14th day, DAPI marked BMSCs were seen in colonic mucosa layer, occasionally seen in the muscular layer and adventitia in 3 groups. On the 7th day and 14th day, the number of marked BMSCs in the mucosa of anabrotic focus of infection was more than those in normal colonic mucosa(F=65.17,P< 0.01). On the 7th day, the number of marked BMSCs of group B in the injured mucosa and normal colonic mucosa were higher than those of group A(F=13.06,P< 0.05); but the difference betwwen group B and group C has no statistical significance.2. SD rats were randomly distributed into D and E groups, and the ulcerative colitis rat model was induced with same method in 2 groups. The suspension of fluorescent-DAPI labeled P3～P5-BMSCs were prepared for cells transplantation. Following the induction of colitis, each rat in 2 groups was received caudal vein injection of 1mL fluids contained 5×106 DAPI marked BMSCs. But in group E, 0.2mg Sodium nitroprusside/kg body weight was injected too. After injection, 5 rats in each group were sacrificed at day 7 and 14, and their colon were prepared as cryostat section. The cell numbers of DAPI marked BMSCs were counted with fluorescent microscope in the colitis tissue and normal colonic tissue respectively. The same section was stained by HE to observe the microscopical changes of pathology.Results: On the 7th day and 14th day, the distribution of DAPI marked BMSCs in group D and E were mainly as same as that in group A,B and C. On the 7th day and 14th day, the number of marked BMSCs in the colonic mucosa of anabrotic focus of infection was more than that in normal colonic mucosa(F=354.54,P< 0.01). On the 7th day, the number of marked BMSCs of group E in the injured mucosa was higher than those in group D(F=15.00,P< 0.05); On the 7th day and 14th day, the number of marked BMSCs of group E in normal colonic tissue was higher than those in group D(F=23.12,P< 0.05)too. Conclusion of whole article1. 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.2. P3-BMSCs can be induced to differentiate into osteogenic cells and adipogenic cells. It demonstrates that the isolated BMSCs have multipotent differentiation capability.3. On the 7th day and 14th day, the number of marked BMSCs in injured colon was more than those in nomal colon. It indicated that the injured tissue and organ may be have specific chemotaxis for BMSCs.4. After vein transplantation of BMSCs with 3 kinds of cell doses into UC rats, the statistical results of BMSCs distributed in colon of UC rats showed that its optimal dose was 5×106 , and not the more of cellular number transplanted in UC rats, the more transplanted cells appeared in the injured colon.5. Comparing between BMSCs transplantation merely groups and BMSCs transplantation united Sodium nitroprusside groups, the cellular number migrated in colon of the former was lower than those of the latter. It indicats that BMSCs transplantation united Sodium nitroprusside could encourage BMSCs migranting to the injured colonic tissue .