| Objective : Bone marrow mesenchymal stem cells (BMSCs) were proliferated and differentiated in vitro. Different dosage glucose culture medium and nicotinamide,Exendin-4 were used to induce the cells into islet-like cells. To verify if the bone marrow mesenchymal stem cells were transdifferentiated into islet-like cells ,we used the immunocytochemical stain,Dithizone (DTZ) stain,transmission electron microscope,radio-immunity analysis . We also wanted to know whether the dosage glucose culture medium and revulsiva have the same effect or not. We have transplanted these induced cells into streptozotocin-induced diabetic rats by caudal vein and renal encyst. The effect of islet-like cells to treat streptozotocin-induced diabetic rats was tested by blood glucose detection.Methods:1 BMSCs from the 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. Cell morphology and growth pattern were observed under invert microscope. The third-generation cells used for the experiment are divided into six groups: 1) low-glucose control group;2) mid-glucose control group;3) high-glucose control group;4) low-glucose induced group;5) mid-glucose induced group;6) high-glucose induced group.2 In order to identify the transdifferentiated cells, we detected them by several methods. Invert microscope was used to investigate the morphologic changes of the cells. Cellular ultrastructure was observed by electron microscope. The insulin-like cells were identified by dithizone (DTZ) and immunocytochemistry. After glucose stimulation, we estimated the induced cells by radio-immunity analysis (RIA).The expression levels of gene about the induced group cells and control group cells were measured by real-time quantitative PCR.3 24 SD rats were introperitonally injected with strep- tozotocin to set up animal model of diabetes and randomly divided into four groups. Each group was consisted 6 rats (3 males and 3 females): 1) renal encyst control group;2) renal encyst group;3) caudal vein control group;4) caudal vein group. The bromodeoxyuridine (Brdu) -labeled cells were transplanted into streptozotocin-induced diabetic rats by kidney capsule and caudal vein. And the clinical effects were observed. Meanwhile, We examined the Brdu-labeled cells distributions in the rats 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 After cultivated by low-glucose,mid-glucose,high- glucose, cell clusters were formed. Addition to nicotinamide,Exendin-4,this trend was obvious .3 Insulin in the induced cells was examined by DTZ stain. To observe the morphology using microscope, the induced group cells were stained and the control group not.4 Insulin secretion of induced cells in every group was detected by immunocytochemical method. With the glucose dosage increasing, the secretion and integral optical density were increased. The result had no statistics difference among different groups(P >0.05). There was significant difference between low- and mid-induced group or high-induced group(P <0.01). Compared mid- with high-induced group, there was no statistics difference(P >0.05).Integral optical density in induced and control groups had distinctness(P <0.01).5 Comparing the non-induced with the transdifferentiated cells ultramicrostructure by electron microscope, some changes were proposed. After being induced the cells showed some typical signs of secretory cell was rich in round or oval-shape cavitation , the mitochondrion in cytoplasm and the structure of rough endoplasmic reticulum was clear , cellular nucleus was unround.6 Insulin secreting was tested by radio-immunity analysis .Each induced group cells could secreted insulin by low- and high-glucose stimulated, and the control group could not. The low-,mid- and high-glucose group were stimulated by low glucose and high glucose, the insulin secreting was increasing(P <0.01).The high glucose was better stimulus than low glucose (P <0.01).7 Real time-PCR quantitative analysisAfter induced by revulsiva,theβ-cell related gene GK,PDX-1,ngn3,GLP-1mRNA expression of the cells were changed. With the additional dosage of glucose GK,PDX-1 mRNA expression were increasing(P <0.01),ngn3 mRNA expression were reducing(P <0.01);GLP-1 mRNA expression were not related to the glucose dosage(P <0.01)Theβ-cell related gene GK,PDX-1,ngn3,GLP-1mRNA expression of the insulin-like cells were changed. With the addition dosage of glucose GK,PDX-1 mRNA expression were increasing(P <0.01),ngn3 mRNA expression were reducing(P <0.01);GLP-1 mRNA expression were not related to the glucose dosage(P <0.01).8 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 renal encyst and caudal vein control group(P >0.05),and the blood glucose were reduced at 7 day and obviously at 14,21,28 day in renal encyst and caudal vein group(P<0.01). And there were no significant difference between renal encyst and caudal vein group(P >0.05).9 The labeling efficiency of Brdu-labeled cells was tested by immunocytochemical stain. The Brdu-labeled cell nucleus were brownish red color,unlabeled were blue. Five high power microscopic (200) sights were selected randomly and observed with light microscope, then the percentage of positive cells (number of positive cells/total number of cells) in each high-power field was recorded. The labeling efficiency was more than 95%.10 We detected the distribution of Brdu-labeled cells in each organ by immunohistochemistry.The Brdu-labeled cells whose insulin-expression positive were tested in the renal encyst group, and the other organ were not found. In caudal vein group, the Brdu-labeled cells with insulin-expression positive were found at lung,liver,kidney,pancreas,but not the heart,spleen,gaster.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 The nicotinamide,Exendin-4 can transdifferentiated BMSCs into islet-like cells. BMSCs were induced in the high-glucose medium and revulsiva are best.3 The transdifferentiated islet-like cells is similar to theβ-cells capability can response to glucose stimulating.4 The transdifferentiated cells could reduced hyperglycemia in the streptozotocin- induced diabetic rats by renal encyst or caudal vein .5 Our research provides a possibility that BMSCs were transdifferentiated in vitro and transplanted those cells to therapy the diabetes in vivo.
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