Bone Marrow Mesenchymal Stem Cells Cocultured With Pancreatic Cells And The Effect On The Hyperglycemia In Streptozotocin-induced Diabetic

Objective: Bone marrow mesenchymal stem cells (BMSCs) were proliferated and differentiated in vitro. Coculturing BMSCs with pancreatic cells transdifferentiated into islet-like cells.To verify if the bone marrow mesenchymal stem cells were transdifferentiated into islet-like cells,we use Dithizone(DTZ) stain and chemiluminescence to detecting its insulin level.We have transplanted these induced cells and MSCs into streptozotocin-induced diabetic rats by caudal vein.The effect of islet-like cells to treat streptozotocin-induced diabetic rats was tested by blood glucose detection and weight.Methods:1 BMSCs from the male SD rats were separated and purified via gradient centrifugation with Percoll solution and adherence to the culture plastic,and then the cells were expanded by subculturing successively.Cells morphylogy and grown pattern were observed under invert microscope.The forth-generation cells used for the experiment are divived into three groups:①PBS group;②BMSCs group;③BMSCs cocultured with pancreatic cells group. 2 The pancreatic cells from the male SD rats were separated and purified via digesting gradually and filtering through a nylon mesh. Then using dithizone(DTZ) stain verify whether the cells were pancreatic cells.3 In order to identify the cocultured BMSCs with pancreatic cells,we detect them by several methods.Invert microscope was used to investigate the morphologic changes of the cells.The insulin-like cells were identified by dithizone(DTZ).After glucose stimulation,we estimated the induced cells by chemiluminescence.The expression levels of pdx-1 about BMSCs and BMSCs cocultured with pancreatic cells were measured by RT-PCR.4 18 SD rats were introperitonally injected with streptozotocin to set up animal model of diabetes and randomly divided into three groups.Each group consisted 6 rats:①PBS group;②BMSCs group;③BMSCs cocultured with pancreatic cells group.The bromodeoxyuridine(Brdu)-labeled cells were transplanted into streptozotocin-inducd diabetic rats by caudal vein.And the clinical effects were observed.Meanwhile,we examined the Brdu-labeled cells distrubitions in the pancreas by immunohistochemistry.Result:1 After 24 hours the inoculation BMSCs could adhere to the flask,where single-dispersal cell and sheet-shaped cell clone were formed,and the cells displayed round,fusiform or multi-angle.The third generation cells were purified and most of them were spindle-shaped.They showed vortex shaped or parallel arrangement growth.2 Pancreatic cells which has entire and clear membrane displayed round or elliptical,and they are not them same size.The cells clusters formed in the first three days.In the forth to seventh days,the cells formed monolayer and assemblage in good condition.After the seventh day,cell debrois were more and more. Insulin in pancreatic cells was examined by DTZ stain.To observe the morphology using microscope,the cells were stained.3 We coculture the purified forth generation BMSCs with pancreatic cells.In the third day,most of the cells were spindle-shaped,very few of them were round or multi-angle.The cells,photonasty enhancd. In the sixth day,most of the cells were round,and formed cell group.The round cells were more in the ninth day.4 Insulin in the induced cells was examined by DTZ stain.To observe the morphology using microscope,the cocultured group cells were stained and the BMSCs not.5 Insulin secreting was tested by chemiluminescence.The induced group cells could secreted insulin by low-and high-glucose stimulated,and BMSCs could not. The high glucose was better stimulus than low glucose(P<0.01).6 We use RT-PCR to detect the expression of pdx-1 mRNA.BMSCs cocultured with pancreatic cells mRNA express in the third day,the expression were increased in the sixth day,and reduced in the fourteenth day. The BMSCs did not express pdx-1.7 On the third day after injection STZ,the rats whose blood glucose≥16.7 mmol/L were lasting 2 days.The model was successful when obviously polydipsia and hyperdiuresis characters.We applied the blood glucose instrument to detect the blood glucose levels by the caudal vein.The blood glucose were not changed in PBS group(P>0.05).The blood glucose were reduced at 21 day in the BMSCs group and BMSCs cocultured with pancreatic cells group.Compared to pretransplant there were significant difference (P<0.01).There were no significant difference between BMSCs and BMSCs cocultured with pancreatic cells group(P>0.05).We could observe the improving of polydipsia,polyphagia and hyperdiuresis characters in the rats,and their fur changed clean,reaction sensitive,pad dryed.8 We detected the distribution of Brdu-labeled cells in pancreas of the rats by immunohistochemistry.The Brdu-labeled cells whose insulin-expression positive were tested in the BMSCs and BMSCs cocultured with pancreatic cells group,and the PBS group were not found.Conclusions:1 It is better that BMSCs are separated and purified by means of gradient centrifugation with Percoll solution and adherence to the culture plastic.2 Via digesting gradually,changing bottle and filtering through a nylon mesh,we could chalk more and pure pancreatic cells.And coculturing to 3 to 7 day,the cells suite to vitro empirical study.3 The transdifferentiated islet-like cells is similar to theβ-cells capability can response to glucose stimulating.4 The transdifferentiated cells could reduced hyperglycemi–a in the streptozotocin-induced diabetic rats by caudal vein.5 BMSCs could reduced hyperglycemia in the streptozotocin-induced diabetic rats by caudal vein.But the effect was not better than the cultured cells.