| ObjectiveIn recent years,Diabetes prevalence rate in the world showing a rising trend,but more younger onset.Stem cells are a group of more primitive cells,they are both self-renewal capacity,is also at a suitable micro-environment to a multi-differentiation potential,in theory,can provide a rich source of islet cells.The research on the differentiation of stem cell into insulin-producing cell provides a new prospect of a cure for DM.In this field,bone marrow stem cell may be one promising kind of stem cells..Bone marrow stem cell have various differentiation potency and conspicuous plasticity.Many vitro studies have successfuly induced the bone marrow stem cell into insulin-producing cell,which could improve hyperglycaemia after transplanted into the body of diadet rat.Accordingly,the present study under conditions of high glucose, Whether or not bone marrow stem cells can be induced in vitro for the IPCs,and to study the differentiation of cells by Nicotinamide united Exendin-4 after the transfusion of diabetes mellitus rats Whether or not there is effective treatment.Material and methods1.BM-MSCs Cell Isolation and inducedRemove the 3-week-old rat femurs under sterile conditions,BMSCs from the rats were separated and purified via direct adherence to the culture plastic.The third-generation cells were detected by flow cytometry and induced to IPCs under following conditions:pre-induction with HG-DMEM+5%fetal calf serum(FCS)for 14 days,release of insulin by these cells confirmed by ELISA.Then added nicotinamide(10mmol/L)to induce for 7 days,and then followed with addition of Exendin-4(10nmol/L)for another 7 days,test the insulin.Cellular ultrastructure was observed by electron microscope.The insulin-like cells were identified by dithizone(DTZ),and then marked the cells with BrdU before translation.2.Animals grouping and treatment28 wistar rats were randomly allocated into two groups:normal group(8),treated group(20,DM group and experimental group),The treated group were treated with single intraperitoneal injection of 1%streptozotocin(STZ,55 mg/kg body wt)to establish diabetes model.The successful 16 wistar rats were randomly allocated into two groups:DM group and experimental group.The marked cells were intraperitoneally injected into diabetic models rats with(5.6-6)×l0~5 differetiated cells. The groups of normal and DM were treated with correspond dose of NaCl.3.IndicationImage acquired at all stages.of cell culture and induction,the induced cells stained by dithizone.In the process of experiment,the weight were measured every week,measured the blood glucose every week before treatment and the end of the study measured the fasting serum insulin concentration at 8 weeks.Treat the rats with intraperitoneal injection of 10%Chloral Hydrate(3 ml/kg body wt)to anesthetize them. remove the liver,spleen,pancreas,and then measure the expression of insulin positive cell by immunohistochemistry,the area of insulin positive cell in pancreatic islet were measured by using image analysis software.Results1.The results of isolation and induce of cellsCell culture 48 hours after there is a small amount of rod-like or polygonal cells adherent growth.After 4 days,the adherent cells were round or rod-like,after 80 percent of cells fused,digestion and.transfer of culture.13 days,we obtain the 3rd generation of homogeneous cells,strong proliferative capacity.After 14 days,the 3rd generation cell culture by HG-DMEM,the cells ability to add value and a strong trend of mission-shaped growth,no significant changes in individual cells.N induced for 7 days and then with the E-induced for another 7 days,individual cells increases markedly,the cytoplasm is rich nuclei become larger,showing polygonal or near round, declining proliferation by DTZ staining cells were stained orange-red.2.Flow cytometryBM-MSCs express CD29 and CD44,the percentage of positive cells were respectively 95.47%and 91.83%;but CD34(hematopoietic stem cell surface specific markers)and CD45(common leukocyte surface antigen)were expressed weakly,the expression of the percentage of cells were respectively 1.05%and 1.18%.3.Biochemical resultsInsulin excreted from differentiated cells by drug was much higher than that from pre-differentiated cells(p＜0.01).Before administration,the blood glucose and fasting serum insulin concentration level has no significant difference among DM group, experimental group.At the end of administration,the blood glucose level of experimental group is higher than the normal group but lower that the DM group(p＜0.05).The fasting serum insulin concentration of experimental group is significant higher than DM group(p＜0.05),compared with normal group,the fasting serum insulin concentration level is much lower(p＜0.01),.Compared with DM group, the blood glucose and fasting serum insulin concentration of experimental group have significant difference.In the whole process,the weight of normal group is always higher than other groups.4.Immunohistochemistry and image analysisAt the end of our experiment we found that the ratio of insulin positive cell in normal group which have been measured by immunohisto chemistry was conspicuous higher than other each group,the ratio of experimental group is lower than normal group,but higher than DM group,and there has significant difference between experimental and DM group.We found insulin and BrdU duble-positive cell expression in pancreas liver and spleen in other groups.ConclusionIPCs can be differentiated from BM-MSCs and translation of these cells can control blood glucose level in STZ-diabetic rats.
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