Study Of Inducing Rabbit Bone Marrow Mesenchymal Stem Cells Into Islet Cells By Alteration In DNA Methylation

Diabetes is one of the major diseases, which has become a serious harm human health. Sofar, islet transplantation is still cure type1diabetes and part of the patients with type2diabetes arethe most effective method. But donor shortage and immune rejection seriously hindered theapplication of islet transplantation. Bone marrow mesenchymal stem cell is a class of adult stemcells of self-proliferation and the multiplex differentiation potential. In the appropriate inducedcondition, it can differentiate into islet cell from germinal layer to endoderm. However, how toefficient and directional differentiation is still a big challenge. Islet cell differentiation mechanismis regulated by a serie of transcription factors in the pancreas development process.DNAmethylation participates in the process as a specific gene regulation so as to participate in stem celldifferentiation. This study is on the basis of optimizing separation culture rabbit BMSCs methodfor researching methylation inhibitors5-aza-dC to islet cell differentiation influence and itsappropriate concentration, and exploreing the level of DNA methylation is correlated with isletcell differentiation,which it is helpful to further research and provide theoretical basis for clinicalapplication.1. Rabbit BMSCs were separated by red blood cell lysis method in this study and BMSCs werecultured by using the principle of full bone marrow method. In order to obtain high purity andvigor strong BMSCs, we optimize culture conditions of cell generation and inoculation densityand immunocytochemistry method identified surface mark. We drew the third generation and theeighth generation of rabbit BMSCs growth curve and calculated group doubling time by cellcounting method2. DNA methylation inhibitors5-aza-dC to the influence of the cell vitality of the rabbit BMSCswas detected by MTT method in this experiment and5-aza-dC appropriate concentration to theeffect of rabbit BMSCs was0.2-1μ mol/L. Rabbit BMSCs were made a previous processing byusing5-aza-dC and it was compared with not adding5-aza-dC induction group by morphology,dithizone dyeing appraisal insulin secretion and immunofluorescence identifying PDX-1protein expression. The results indicated that the use of DNA methylation inhibitors5-aza-dC, DMSO,niacinamide and high sugar induction of BMSCs have the ability to secrete insulin and expressingthe specific makers of islet cells. The methylation inhibitors5-aza-dC can promote islet celldifferentiation and maturation.3. On the basis of second experiment, RNA was extracted from induced islet cell and then wasreverse transcription into cDNA. PCR was amplified by using the design of primers.We detectedan important regulation factor PDX-1gene by gel electrophoresis and observed results underultraviolet projection instrument.Conclusion: DNA methylation inhibitors5-aza-dC in combination with DMSO, niacinamide,and high sugar can induce rabbit BMSCs into insulin-producing islet cells. Methylation wasinvolved in regulation islet cell differentiation and low methylation promoted islet specific geneexpression. Moreover, it further promoted the islet cell differentiation and maturation.