The Study Of Rabbit Bone Marrow Mesenchymal Stem Cells Differentiating Into Islet Cells In Vitro

Diabetes is a chronic metabolic disease which is severely impair humanity health and charactered as hyperglycosemia.Islet transplantation is one of the most promising methods for the treatment of diabetes,but the function of islets will gradually disappear in the sixth months to a few years after islets transplantation and it face with the problem of lack of donor and immune rejection.Bone marrow mesenchymal stem cells(BMSCs)are another kind of adult stem cells with the capability of high proliferation and self-renewing and potentials of multiple differentiation in the bone marrow in addition to hematopoietic stem cells,they are easy to isolate,culture and amplify invitro,they can be differentiated into insulin cells under certain conditions and avoid the theoretical problems involved in the use of embryonic stem cells and the technical safe problems arising from the induced pluripotent stem cells,so they can be used as an ideal tissue-engineering seed cells.However,it is a huge challenge that how to make rabbit BMSCs differentiate into insulin cells stably and efficiently and clarified the molecular regulation mechanism of BMSCs differentiation into insulin cells in related induction method.In this study,we used one-month-old Japanese big-ear rabbits as the experimental object,isolated,cultured and amplified rabbit BMSCs,observed the morphological changes of rabbit BMSCs,identified rabbit BMSCs by immunohistochemical staining,established a stable induction method of rabbit BMSCs differentiating into insulin cells and analysed the molecular regulation mechanism of rabbit BMSCs differentiating into insulin cells in vitro,explored the optimal intervention concentration of DNA methylation inhibitor 5-aza-d C on rabbit BMSCs and its effects on the differentiation into islet cells,and our study provided the theoretical basis and technical support for the further exploration of more mature induction method and BMSCs replacement therapy for diabetes.1.In this study,rabbit BMSCs isolated by the whole bone marrow adherent method had a strong proliferative ability and stable biological character.For the isolated bone marrow cells,we changed half medium firstly after 24 hours,then changed the medium every two days,and the cells were passaged to the third generation by 1:2.P3 rabbit BMSCs were identified by immunohistochemical staining.The results showed that CD44 and CD90 was positively expressed,CD34 was negatively expressed.It fit in with the characteristics of BMSCs surface marker,and we could get alot of rabbit BMSCs with good growth condition and strong proliferation ability.This showed that the obtained P3 cells were just rabbit BMSCs,the method for isolation and purification we used was an ideal method for isolation and purification of rabbit BMScs,and we could choose the third generation of rabbit BMSCs as the experimental object of the following studies,it layed the foundation for the following induction experiment.2.The study inducted rabbit BMSCs at the third generation with Dimethyl Sulphoxide(DMSO)combined with high glucose in two phases,the cell morphological changes of the induction group and the control group were observed under the inverted microscope,the inducted cells were identified by dithizon staining,the expression of insulin protein was identified by immunofluorescence staining,and the expression of related genes of islet cells was detected by RT-q PCR method.Conclusions were as follows:(1)Foxa2,Nestin,Pax6 genes could not be chosen as the marker genes of rabbit BMSCs differentiating into islet cells successfully.(2)DMSO combined with high glucose could induce rabbit BMSCs into islet-like cells with insulin secreting function,and rabbit BMSCs had the potential to differentiate into the endodermal cells.(3)DMSO could active the expression of Pdx-1 gene so that it had the function of promoting the differentiation of rabbit BMSCs into islet precursor cells that could secrete insulin.(4)High glucose could promote the maturation of islet precursor cells and the differentiation of rabbit BMSCs into islet cells,and the promoting effects might be accomplished mainly through promoting the expression of Pdx-1 and Foxa2 gene.3.In this study,MTT method was used to detect the effects of different concentrations of 5-aza-dc on the proliferation ability of rabbit BMSCs in vitro for 24 hours and the optimal intervention concentration was determined.Rabbit BMSCs were intervened by 5-aza-dc at the optimal intervention concentration for 24 hours,then were inducted with DMSO combined with high glucose,the cell morphological changes of the induction group and the control group were observed under the inverted microscope,the inducted cells were identified by dithizon staining,the expression of insulin protein was identified by immunofluorescence staining,and the effects of 5-aza-dc on the expression of related genes of islet cells were detected by RT-q PCR method.Conclusions were as follows:(1)The optimal concentration of 5-aza-d C for rabbit BMSCs was 1.5 umol/L.(2)5-aza-dc could promote the maturation of islet precursor cells and the differentiation of rabbit BMSCs into islet cells,and the promoting effects might be realized by the activation,transcription and increased expression of Pax6,Pdx-1 and Insulin gene caused by Pax6,Pdx-1 and Insulin gene demethylation which was caused by inhibiting the activity of DNA methyltransferase.(3)The low degree of methylation could promote the expression of Pax6,Pdx-1 and Insulin gene.