| Objectives1.Observation of gastrin and epidermal growth factor in vivo induction of bone marrow mesenchymal stem cells into insulin-producing cells differentiation capacity.2.Induced differentiation of insulin-producing cells insulin secretion and therapeutic effectsMethods1.Extraction of rat femur bone marrow-derived mesenchymal cells in vitro passage.The 3rd generation cell was analysed by flow cytometry for identification of bone marrow-derived mesenchymal stem cells.Then it marked by BrdU to be induced in vivo transfusion.2.32 wistar rats were randomly allocated into normal group,diabetes group,stem cell transfusion group and stem cell transfusion-induced group(8 wistar rats in each group).The last three groups of rats were treated with single intraperitoneal injection of 2%streptozotocin(STZ,50 mg/kg body wt)to establish diabetes model.3.After blood glucose stability,the transfusion group and transfusion-induced group in vivo transfusion 3rd generation of bone marrow mesenchymal stem cells, while at the same time transfusion-induced group induced by intraperitoneal injection 3ug/mlGastrin(3ug/kg.d)and 3ug/mlEGF(1ug/kg.d),for 14 consecutive days,the normal group and diabetic group and transfusion group at the same time a considerable dose of intraperitoneal injection of saline.Administration after the end of observation rats were killed after 2 weeks.4.Experiment measured weight weekly,every two weeks a blood glucose measurement.Before treatment and the end of the study measured the fasting serum insulin concentration,at the end of the study,treat the rats with intraperitoneal injection of 10%chloral hydrate(3 ml/kg body wt)to anesthetize rats,and remove the liver, spleen,pancreas and then measure the expression of BrdU/insulin staining double positive cells by immunohistochemistry.By the Image Acquisition and Analysis System of the Group of pancreatic insulin-positive cells staining regions accounted for the percentage of islet area for analysis.Results1.The results of flow cytometry The Cultured BM-MSCs express CD29,CD44 positive,CD34,CD45 negative.2.Biochemical test results Before administration,the blood glucose and fasting serum insulin concentration level has no significant difference among DM group,the bone marrow transfusion group and transfusion-induced group.At the end of administration,the blood glucose level of bone marrow transfusion group and transfusion-induced group is higher than the normal group(P＜0.01)but lower that the DM group(P＜0.01).At the end of study,the blood glucose level of transfusion-induced group is significant lower than DM group(P＜0.01),but still higher than normal group, the fasting serum insulin concentration of transfusion-induced group is significant higher than DM group(P＜0.01),compared with normal group,the fasting serum insulin concentration level is significant lower than normal group.Compared with DM group,the blood glucose and fasting serum insulin concentration of transfusion group have no significant difference.3.Immunohistochemistry and image analysis results At 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(P＜0.01),the ratio of transfusion-induced group is lower than normal group,but higher than DM group and transfusion group(P＜0.01),and there has no significant difference between transfusion and DM group(P＞0.05).In transfusion-induced group We found BrdU staining/insulin staining double positive cells expression in liver and pancreas,but no expression in spleen.We did not find any expression of BrdU staining /insulin staining double positive cells in others liver and spleen.Conclusion1.Combination therapy with Epidermal Growth Factor and Gastrin could induce differention of bone marrow mesenchymal stem cell into insulin-producing cell in vivo.2.Insulin-producing cells from Bone marrow mesenchymal Stem Cell have certain effect on rat diabetes.
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