| Objective:1. To establish one method of isolation and cultivation of Transgenic C57BL/6 male mice BMSCs in vitro. 2. To study the capacity of allogeneic bone marrow mesenchymal stem cells (BMSCs) survival and homing in ischemic muscular pouches in the same gene mice.Methods:1. The isolation culture and purity identification of EGFP-BMSCs: bone marrow cells of EGFP-mice Adherent culture in the culture bottle to cultivate. By way of changing the culture medium gradually, the cells which didn't Adherent to the wall may being taken away and EGFP-BMSCs obtain purification and detect the purity of the fifth passage EGFP-BMSCs by flow cytometry.2.Animals group and model established:Animals were divided into group A.B.C at random. Group A(Quadriceps muscle pouch model of ischemia and local injection group):we make ischemic muscular pouches-of the right quadriceps in mice (dissociate quadriceps, ligation of the near and far side,the same below), then injected about 0.01ml EGFP-BMSCs into the right ischemic muscular pouches of 30 C57BL/6 mices as allograft model(the same below), the same muscle pouches on the left was injected 0.01ml isotonic Na chloride as muscle control group; Group B(Normal quadriceps muscle pouch model of local injection group):exposed Bilateral quadriceps in mice, then injected about 0.01ml EGFP-BMSCs into the right muscular pouches of 30 C57BL/6 mices as allograft model, the same muscle pouches on the left was injected 0.01ml isotonic Na chloride as muscle control group; Group C(Quadriceps muscle pouch model of ischemia were injected group by tail vein): exposed Bilateral quadriceps in mice, make ischemic muscular pouches of the right quadriceps in mice, then injected about 0.01ml EGFP-BMSCs into 30 C57BL/6 mices by tail vein, the right muscular pouches as allograft model;3. Killed 18 mice of each group respectively after the first day, four days, one week, two weeks, four weeks of operation, and then obtain both muscular pouches of each mice, Extracted muscular pouches protein at different time and make Western-blot test, to calculate the gray scale ratio of bands, and make statistical analysis.Results:1. Apply the method of different speed of adherence,change culture medium and passage to isolate and culture EGFP-BMSCs can obtain enough quantity of EGFP-BMSCs.2.After allogenic transplantation of EGFP-BMSCs, there were not obviously abnormal reaction at the host C57BL/6 mice.3.After allograft transplantation of A.B.C groupg, we finding that the EGFP-BMSCs can be survived at least four weeks which can be detected by western-blot, the amount of EGFP gradually reduced with time4.All the normal sides did not detect the EGFP.5. Statistical analysis:use Quantity one software to analyze Western blotting bands of EGFP at different times of ischemia injection group, normal injection group of local and systemic injection group, all the data obtained for statistical analysis by the SPSS 16.0 statistical software.Conclusion:1. BMSCs could be isolated and proliferated effectively by whole bone marrow adherence method.2. Can successfully constructed ischemia model by ligation of the quadriceps muscle pouch.3.Host did not have acute or obvious rejection performance after allograft transplantation of EGFP-BMSCs.4.the allogeneic EGFP-BMSCs can survive more than 4 weeks in the local muscle pouches of host by local injection; EGFP-BMSCs by local injection have the phenomenon of local enrichment which can be used as a feasible approach for stem cell therapy.5. Intravenous injection (systemic injection) EGFP-BMSCs could through the non-vascular way homing to ischemic injury in the host muscle pouch,which can survive more than 4 weeks, but the amount of EGFP-BMSCs gradually reduce with time.6. EGFP-BMSCs of Homing to the ischemic mouse muscle pouches might be involved in damage repair and the formation of blood vessels... |
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