The Study Of Inducing Rabbit Bone Marrow Mesenchymal Stem Cells Into Islet Cells

Bone marrow mesenchymal stem cells(BMSCs) is a kind of multiplex differentiation potential of stem cells.lt originates from mesoblast, resides mainly in mammalian bone marrow, and coexists with hematopoietic stem cells(HSCs). BMSCs not only participation in the construction hematopoietic microenvironment, give play to the role of hematopoietic support, still have the cross into a variety of cell types including endocrine cells. Because of its easy to separate, trainand amplified in vitro. BMSCs with undifferentiated or not genetically modified is expected to become ideal seed cells for autologous transplantation, gene therapy, tissue engineering technology in future. This study aims to purification rabbit BMSCs. and study its potential of differentiation into islet cells in vitro, so that it can be used as a disease cells and gene therapy model in future.Therefore, the test uses a large white rabbit bitching New Zealand as the research object, relatively systematically study the BMSCs separation, training, amplification, identification, morphology and the capacity of differentiation in vitro. and lay a test basis for the further research BMSCs of differentiation and application.1.This study applies the separation method of the bone marrow sticking to wall, according to the bone marrow stemL cells, growth characteristics training in the bottle posted wall.successfully separated rabbit BMSCs. purify BMSCs by continuous passage culture.When culturing to the third generation in vitro, get high purity of BMSCs. This study conducted morphological observation and Immunocellulerchemistry for the third generation purification cells, concluded that the rabbit BMSCs surface was positive with CD90CD44, negative with CD34, confirmed that the purification cells just was rabbit.BMSCs and layed the foundation for further study.2.The study induced BMSCs at third generation with dimethyl sulfoxide (DMSO) and high concentration of glucose. After induced7days,the cells appearred groups of cells. After10days, groups of cells got closely, whereas the control cells did not appeare group of cells always. After treated with dithizone(DTZ) and Immunocellulerchemistry (ICC), the induced cells both were positive and the control group both were negative.In ELISA tests,the insulin content of induced group with (27.82±0.43) mU/L,(27.89±0.25) mU/L, of7d,10days were greatly significant difference (P <0.01) comparing with the control group (23.39±0.20)mU/L (25.71±0.27)mU/L. The result indicates that DMSO with high concentration of glucose can induce rabbit BMSCs into islet cells in a relatively short period.3.The study induced BMSCs in DMEM/F-12with l0ng/mLbFGF、10ng/mLEGF、2%B27for6d,then continued to induce BMSCs in H-DMEM with l0ng/mLHGF、l0ng/mLBTC lOng/mLActivineA、l0mmol/LNicotinamide,2%B27for6d.After12d,BMSCs in treatment group shrinkaged and mutually gathered into round group of cells.while there was not round group of cells in the control group. Through double dithizone(DTZ) dyeing.the reatment group was positive.while the control group was negative,showed that the treatment cells have the typical character of islet cells bing rich in zinc ion. Through immunocytochemistry.the reatment group was positive,while the control group was negative, show that the BMSCs after induction has the ability to secrete insulin. The result showed that cells group of induction in this study are islet cells. The results show that, cell growth factor joint role can differentiate BMSCs into islet cell.4.The study divided7groups with group a、b、c、d、e、f、g. among them a group as control group was cultured in DMEM/f-12culture medium containing10%of FBS. Group b was first cultured in DMEM/f-12culture medium with no serum, containing1%of DMSO for3d. then reoccupy in H-DMEM medium for stimulation training. C, d, e, f groups were respectively added with Nickamide of10mmol/L on1.3.7.10d.in the basis of group b. After induction, b. c. d. e and f groups all had islet cells group appearring. and double dithizone dyeing were positive. Group g had a mild cells together group phenomenon, double dithizone dyeing has a weak positive. Group a had not seen the group of cells, and double dithizone dyeing was negative.ELISA test results showed that Nickamide has the role of promoting bone marrow mesenchymal stem cells to secrete insulin, but still cannot independently differiatiate BMSCs into islet cells. Nickamide can be used together with DMSO to promote islet ceils of differentiation to secrete more insulin with no influence of the adding time.