The Study Of Pancreatic Beta-cells Derived From Bone Marrow Mesenchymal Stem Cell

Background/Aims Bone marrow stem cells have been shown to differentiate into fibroblasts, skeletal muscle, os, cartilage et al. It has also been confirmed that mesenchymal stem cells (MSCs) can be induced into chondrocytes. MSCs may be obtained from different kinds of tissues such as bone marrow, adipose and muscle etc.. MSCs derived from bone marrow are named bone marrow stem cells (BMSCs). Because of their enough resource, less lesion when harvesting, simple culture methods, and high proliferative capability, BMSCs have become the ideal seed cells for cell transplantation therapy. We hypothetical that if pancreatic beta-cells could also derive from bone marrow cells, not only we can confirm the plasticity of stem cells, but also and the most important thing is we can easily obtain pancreatic cells from bone marrow and can use self bone marrow stem cells transplantation to treat patients who need pancreas or pancreatic island transplantation. The great advantage of this cell transplantation therapy is not require immunosuppression, so this approach appears to be highly advantageous for clinical use. Our research aims at solving following problems: (1) To clarify the influences of inductive factors and coculture with pancreatic island cells on BMSCs differentiation; (2) To testify the differentiation of BMSCs in pancreas; (3) To explore the effect of transplantation of BMSCs on diabetic rats. 1. In vitro derivation of pancreatic endocrine cells from bone marrow mesenchymal stem cells Objective: To clarify the influences of inductive factors and coculture with pancreatic island cells on BMSCs differentiation. Methods: Isolation and primary culture of BMSCs from the thighbone of male SD rats were performed according to Wakitani's method. The BMSCs induced with basic fibroblast growth factor (bFGF) , Nicotinamide and transforming growth factor beta-1 (TGFβ). We also coculture the BMSCs with pancreatic island cells, the two kinds of cells did not contact directly but the culture medium could exchange freely. The effects of these factors on differentiation of BMSCs were observed by immunohistochemistry, RT-PCR and cell growth morphology change. Results: BMSCs induced by TGFβexpressed pancreas specific genes such as insulin and Pdx-1, while the expression of Glucagon and Somatostatin mRNA was negative. And insulin could also be detected by immunohistochemistry. While adding bFGF or nicotinamide these expression could not be detected. BMSCs cocultured with pancreatic island cells, except its morphologychanged like pancreatic island cells, the other expression was negative. Conclusion: In the presence of TGFβin vitro, BMSCs could be differentiated into pancreatic beta-cell like cells. 2. The differentiation of bone marrow mesenchymal stem cells in pancreas Objective: To testify the differentiation of BMSCs in pancreas. Methods: We dyed BMSCs with Hoechst 33258 which could combine with DNA, then the cells could give out blue fluorescent. We transplanted dyed BMSCs into pancreas directly to explore its differentiation in vivo. After transplantation, the whole pancreas was removed from the rats every three days. The pancreas slices were examined under fluorescent microscope to detect blue fluorescent positive cells. Insulin was detected on the same slices by immunohitochemistry. Results: The transplanted blue fluorescent positive BMSCs were found in rats'pancreas for more than one month. Though immunohistochemistry, the transplanted BMSCs seemed to retain insulin-producing ability. From morphology, we detected that BMSCs also differentiated into blood vessel forming cells. Conclusion: BMSCs could be differentiated into pancreatic beta-cell like cells and blood vessel forming cells. 3. Effect of transplantation of bone marrow mesenchymal stem cells on diabetic rats Objective:To study the effect of transplantation of bone marrow mesenchyml stem cells on diabetic rats. Methods:The diabetic rat model was induced with a single intraperitoneal injection of streptozotocin. The diabetic rats were divided into two groups, control group and transplanted group. The control group was intraperitoneal injected same volumeculture medium. The transplanted group was directly injected the bone marrow mesenchyml stem cells into pancreas. The blood glucose level was evaluated . Results:After transplantation blood glucose level of diabetic rats was decreased significantly from 17.58±2.46mmol/L to 5.94±2.25mmol/L (P<0.01). The blood glucose level between control group and transplanted group was significantly (P<0.01) .Conclusion:The diabetic rats'blood glucose level was lowered by bone marrow mesenchyml stem cell transplantation.