Bone Marrow Derived Mesenchymal Stem Cells Promote The Regeneration Of Islets

IntroductionDiabetes with the main character of high blood glucose concentration is considered to be a severe disease of human being. Diabetes causes about 5% of all deaths globally each year. Diabetes deaths are likely to increase by more than 50% in the next 10 years without urgent action. The type I diabetes is due to the decrease of absolute quantities of islet. Islet transplantation becomes an effective method for diabetic therapy. However, the immune rejection and the deficiency of the donor are big obstacles for this method.Stem cells are multiclonal cells capable of both self-renewal and multilineage differentiation into any type of cell and to be genetically modified in vitro, thus providing cells which can be isolated and used for transplantation. Inducing the ES cells in to the insulin-secreting cell cluster in vitro is another direction for the diabetes therapy. But the problems are the low induction ratio, the complex induction process, the immune rejection and the easiness of tumor formation. Many technologies need to be resolved in order for the clinical application. Bone marrow mesenchymal stem cells (MSCs) are adult stem cells isolated from the adult bone marrow, which possess an extensive proliferative potential and ability to differentiate into various cell types,such as osteocytes, chondrocytes and myocyte et al. MSCs is easily available, bears low immune reactivity, can be expanded largely in vitro without alteration of genetic characters. Transdifferention of MSCs into insulin secreting cell clusters has great clinical application prospect. However, it also has the problems like low differentiation ratio. It was reported transplanting bone marrow can mitigate the diabetic syndromes and reduce the high blood glucose. The mechanism still needs to be elucidated. Hess reported that mouse bone marrow derived stem cells (c-kit+) can initiate the regeneration of the endogenous islet. They thought the donor derived endothelial cells (PECAM+) participated in the regeneration. However, the mechanism that which cell from the bone marrow contribute, and in which way to reduce the blood glucose still to be elucidated. Allogenic transplantation of mouse and rat MSCs can repair many tissue injures, such as myocardial ischemia, kidney, lung injures et al. But the mechanism still requires being uncovered. Some reports indicated that the new 0 cells were formed from the existingβcells rather than from the differentiation of the stem cells. It has not been reported whether MSCs transplantation alleviating symptoms of high blood glucose of diabetes was relative to the proliferation of islet.Cdk4 gene is thought to be key cell cycle protein to regulate the cell proliferation. It can direct the entrance to the S phase from G1 phase by forming the complex with cyclin D. Cdk4 is reported to regulate the regeneration, proliferation and senesce of isletβcells. So whether MSCs alleviate the high glucose syndromes of the STZ induced diabetes though the pathway of promoting the regeneration of islet needs to be elucidated.It was reported that human and mouse islets can dedifferentiate into pancreatic precursor cells in vitro. While REST gene did not express in mature neural cells and islet cells. The mechanism of the islet proliferation via the dedifferentiation to REST positive cells stage remains to be elucidated. Whether MSCs promote the regeneration of islet cells by this way to alleviate symptoms of high blood glucose of diabetes is not clear.We cocultured the pancreas lysate and MSCs in vitro. MSCs could be induced to be islet-like cells. MSCs were transplanted into the diabetic rat and observed the decreases of the blood glucose and improvement of diabetes syndroms. The aim of our study is to explore the potential mechanism of MSCs to promote the regeneration of the isletβcells both in vitro and in vivo and the Cdk4 and REST gene expression levels.Method1,Isolation and identification of MSC in vitro(1) The tibias and fetuses of rats are removed aseptically for the primary culture.(2) Sub-culture the primary MSCs to get the homologous cells.(3) Identify the expressions of CD14, CD34, CD44, CD45, CD73, CD90 of the MSCs by flow cytometry methods2,Lentivirus transfection of MSCs(1)Package the recombinant lentivirus plamids pGCL-GFP(2)Lentivirus transfection of the MSCs3,Establishment of the diabetic rat model and MSCs transplantation(1) Establishment of the diabetic rat model by intraperitoneal injection of Streptozocin(2) Glucose detection to validate the successful establishment of the diabetic rat model(3) Caudal veins transplantation of the transfected MSCs(4) Detection of the changes of the glucose concentration after transplantation4,Detection Islet squares by immunocytochemistry and Statistical analysis(1) Obtain the cryosections of rat pancreas(2) Value the square of the islet by insulin immunofluorescence(3) Use the software to analyze the differences among groups5,Coculture of MSCs and insulin secreting cell line INS1, islets(1) Culture the MSCs and INS1, islet in separate room of one transwell(2)Detect the expression of cell proliferation gene Cdk4 and REST by real-time PCR Results1,The rat bone marrow mesenchymal stem cells were isolated and identified the expression of CD14(-), CD34(-), CD45(-), CD44(+), CD73(+), CD90(+) by flow cytometry.2,The blood glucose concentration of the transplantation group was significantly lower than the control group 11 days after cell transplantation. However, the blood glucose concentration elevated after then without significant difference compared to the control group.3,Transplanted MSCs engrafted the pancreas, without expression of insulin4,The immunofluorescence square from the transplanting group was significantly larger than the control group. The injured islet was promoted to regenerated5,The Cdk4 gene of INS1 cell was high expressed by 2.281 and 1.869 folds respectively after coculturing the MSCs with INS1, islets.6,The REST gene of islet was high expressed by 1.608 after coculturing the MSCs with islets.Conclusion1,Transplanting the bone-marrow derived mesenchymal stem cells can alleviate the high blood glucose syndromes of STZ induced diabetis in the short periods.2,The Cdk4 and REST genes were high expressed after coculturing the islets with MSCs.