| Background The diabetes mellitus is a chronic metabolic disease which influences the health of human being seriously. The number of patients suffer from diabetes continue to increase all over the world. Both type1and type2diabetes result from an inadequate mass of functioning beta cells, and thus, beta cell regeneration has been providing new therapeutic approaches for diabetes. Many studies have made efforts to expand pancreatic islets and to develop renewable sources of islet-replacement tissue. Researches indicated that transplantation of human umbilical cord mesenchymal stem cells which have great potentialities of the continuous proliferation and differentiation promotes islets repair in diabetic rat. Therefore, it is thought to be a valuable source for generation of beta cells. However, transplanted site provides a necessary microenvironment for graft survival and function exertive. It’s when the graft plants steadily and survives chronically that transplantation will succeed. At present, the ways of stem cells transplantation in vivo are varied and the effects are different, there is no consistent conclusion about the most ideal transplanted site. In addition, the therapeutic effects of hUCMSCs have been approved generally, but its hypoglycemic mechanism is still not yet clear, and controversy persists regarding whether the hUCMSCs can transdifferentiate into beta cells.Objectives To establish type1diabetes mellitus rat model with characteristics of beta cells destruction and to observe the hypoglycemic effects of hUCMSCs transplantation in different sites. Try to explore the most ideal transplanted site. To detect the mRNA expression of murine&human insulin, PDX-1and nestin in the recipient’s pancreas6weeks post-transplantation. Try to investigate whether hUCMSCs transdifferentiate into beta cells and to explore the mechanism of hUCMSCs repair the damaged islet.Methods (1)70SPF male Sprague-Dawley rats,7weeks old, weighting about230-250g, were housed in an environmentally controlled room and after two weeks adaptive feeding, they were randomly divided into two groups:the normal control group(NC group, n=10) and the Streptozotocin group(STZ group, n=60). After fasting for12hours, Streptozotocin group rats were intraperitoneal injected with Streptozotocin (STZ,70mg/kg). We dtected the random blood glucose before injection and after intraperitoneal injection with STZ for48h,5d,8d,11d,14d. The rats whose random blood glucose was continuously more than16.7mmol/l were identified as diabetes mellitus models and whose random blood glucose was less than16.7mmol/l were excluded.(2) Then the type1diabetic model rats were divided randomly into four groups: diabetic model control group (DMC group, n=14), pancreatic subcapsular place transplantation (Pan group, n=14), nephritic subcapsular place transplantation (Nep group, n=14), intrahepatic parenchyma transplantation (Hep group, n=14). After given intraperitoneal anesthesia,1×106human umbilical cord mesenchymal stem cells per rat were injected into diabetic rat’s pancreatic subcapsular place, renal subcapsular place and intrahepatic parenchyma respectively. The normal control group and diabetic model control group were injected with normal saline solution into pancreatic subcapsular place. Body weight, fasting blood glucose and fasting insulin level were measured weekly after transplantation.(3) Six weeks post-transplantation, after given intraperitoneal anesthesia, the rats’ pancreas, kidney, liver tissue was collected to observe histological changes by HE stain and to characterize the pancreatic histology, morphology and protein expression of rat&human insulin by histochemistry. And the pancreas tissue were collected in liquid nitrogen to examine the mRNA expression of murine&human insulin, PDX-1and nestin by reverse transcription-polymerase chain reaction (RT-PCR).(4) Then analysed the correlation of these data. All data were reported by the way of "mean±standard deviation". SPSS13.0statistics software was adopted for analysis of these data. P<0.05was considered statistically significant difference.Results (1) Compared with NC group, the random blood glucose level of STZ group rats increased after injection of STZ with significant statistic difference (P<0.05).(2) The operative procedure went smoothly, and the wound healing, rats survived well after operation. To the end of the observation period, the rats of NC group all survived, the mortality rate of DMC group, Pan group, Nep group, Hep group were28.6%,21.4%,21.4%, and28.6%.(3) After transplantation, the body weight levels of DMC group、Pan group、Nep group and Hep group rats lasted to the end of the experiment were lower than that of the NC group with significant statistic difference (P<0.05). Compared with the DMC group, the body weight levels of Pan group were significantly increased at three weeks after transplantation (P<0.05) and sustained to increase at six weeks after transplantation (P<0.05)After transplantation, the FBG levels of DMC group、Pan group、Nep group and Hep group rats lasted to the end of the experiment were higher than that of the NC group with significant statistic difference (P<0.05), and the FINS levels were lower with significant statistic difference (P<0.05). Compared with the DMC group, the FBG levels of Pan group were significantly reduced at two weeks after transplantation (P<0.05), and sustained to reduce at six weeks after transplantation (P<0.05). Compared with the DMC group, the FINS levels of Pan group were significantly increased at two weeks after transplantation (P<0.05), and sustained to increase at six weeks after transplantation (P<0.05) Compared with the DMC group, the FBG levels of Nep group were significantly reduced at three weeks after transplantation (P<0.05), and sustained to reduce at five weeks after transplantation (P<0.05); Since sixth week, the FBG levels of Nep group increased again, and there were no statistic difference between the two groups (P>0.05). Compared with the DMC group, the FINS levels of Nep group were significantly increased at three weeks after transplantation (P<0.05), and sustained to increase at five weeks after transplantation (P<0.05); Since sixth week, the FINS levels of Nep group reduced again, and there were no statistic difference between the two groups (P>0.05). Compared with the DMC group, the FBG levels of Hep group were significantly reduced at two weeks after transplantation (P<0.05), and sustained to reduce at four weeks after transplantation (P<0.05); Since fifth week, the FBG levels of Hep group increased again, and there were no statistic difference between the two groups (P>0.05). Compared with the DMC group, the FINS levels of Hep group were significantly increased at two weeks after transplantation (P<0.05), and sustained to increase at four weeks after transplantation (P<0.05); Since fifth week, the FINS levels of Hep group reduced again, and there were no statistic difference between the two groups (P>0.05).(4) Pathology detection:No exogenous islet-like cell clusters were found except the Pan group recipient’s pancreas6weeks post-transplantation. The exogenous islet-like clusters expressed rat insulin.Immunohistochemistry detection:Compared with NC group, the ratio of the average optical density of rat insulin in DMC group, Pan group, Nep group and Hep group were significantly decreased(P<0.05). Compared with the DMC group, the level of rat insulin in Pan group was significantly increased (P<0.05). Compared with the DMC group, the levels of rat insulin in Nep group and Hep group had no significant statistic difference. There was no human insulin stain in all tissue slice.(5) RT-PCR:There were no human insulin mRNA expression in NC group, DMC group and Pan group, so were human PDX-1&nestin mRNA expression. The mRNA expression of rat insulin in Pan group was lower than that in NC group with significant statistic difference (P<0.05), but was higher than that in DMC group with significant statistic difference (P<0.05). The mRNA expression of rat PDX-1&nestin in Pan group were higher than that in NC group with significant statistic difference (P<0.05), and were higher than that in DMC group with significant statistic difference (P<0.05). Conclusions1. Given high dose STZ by intraperitoneal injection, the stable type1diabetes mellitus SD rat model can be established successfully.2. hUCMSCs transplantation can efficiently treat diabetic rat and the ideal transplanted site was pancreas.3. hUCMSCs can’t transdifferentiate into pancreatic islets β cells in diabetic rat. hUCMSCs transplantation can initiate pancreatic islets β cells regeneration, proliferation and differentiation of pancreatic stem cells may contribute to the regeneration of β cell. |
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