| Objective1. Establishing the high-sugar-fat diet add streptozotocin (STZ) injection induced diabetic nephropathy model of the tree shrew and evaluation methods.2. To observe in vitro DAPI labeled tree shrew bone marrow mesenchymal stem cells (BMSCs) method and therapeutic effect of transplantation of cells to diabetic nephropathy in tree shrew, providing the theory basis and reference method for stem cell therapy for diabetes.Method1.Establish the tree shrew model of diabetic nephropathy:using high-sugar-fat diet at the same time giving fasting intraperitoneal injection of streptozotocin (STZ100mg/kg) method to establish the tree shrew model of diabetic nephropathy. Detection of triglyceride, total cholesterol, blood creatinine, urea, insulin, glycosylated hemoglobin,24h urine protein, urine volume, body weight and24h oral glucose tolerance test (OGTT) of tree shrew, to evaluate model. Further analysis using HE staining, observation of pathological features of pancreas and kidney, the tree shrew model reached basic standard with diabetic nephropathy. The tree shrew model with the typical characteristics of diabetic nephropathy were selected to further experimental research.2.1solation and identification and labeling of the tree shrews mesenchymal stem cells:tree shrew femoral and tibial bone marrow cells were collected under sterile conditions, with reference to the previous established human and other animal bone marrow mesenchymal stem cells culture method were primary cultured and passaged in vitro. Using flow cytometry in tree shrews with bone marrow mesenchymal stem cell surface antigen CD90, CD105, CD31, CD34expression, observation of positive expression rate of bone marrow mesenchymal stem cell specific antigen. For the tracking of the distribution and evolution of transplanted cells in model body. Method for DAPI labeled tree shrew BMSCs in vitro was used in this experiment, the labeling rate was detected by fluorescence microscope.3.The tree shrew BMSCs transplantation and the efficacy analysis after transplant: the tree shrew of DN+MSC group were injected DAPI labeled BMSC through the tail vein each tree shrew was injected about5x106cell/kg. The control group and DN group were injected with saline. Changes of the tree shrews peripheral blood glucose, triglycerides, total cholesterol, creatinine, urea, insulin,24h urine volume,24h urine protein were detected by using automatic biochemical analyzer after BMSCs transplantation.The pancreas and kidney were fixed by4%paraformaldehyde and made tissue sections. According to conventional methods, HE staining was procede to observe the change of pathological tissue structure under light microscope and photographed. The pancreas, kidney tissue were made frozen section and to observe the distribution of transplanted cells in tissues by fluorescence microscopy.4.Statistical methods:all the data were statistically analyzed by SPSS17.0statistical software, the measurement data were with mean and standard deviation (x±s), statistical differences between groups were compared with single factor analysis of variance (One-Way ANOVA). P<0.05indicated a statistically significant difference.Result1.The tree shrews diabetic nephropathy model index:the tree shrew of model group appeared polydipsia, polyuria and other clinical symptoms. During the experiment, blood glucose was significantly higher (23.59±1.37vs.3.56±0.76, P <0.01), triglycerides (1.25±0.2vs.0.59±0.11, P<0.01), total cholesterol (7.46±0.79vs.2.84±0.24, P<0.01), creatinine (43.25±7.25vs.19.25±3.3, P <0.01), urea (15.75±2.83vs.6.00±0.26, P<0.01),24h urine protein (4.74±1.62vs.1.38±0.86,P<0.01) increased significantly;24h urine volume increased significantly (18.56±3.50vs.3.47±1.29, P<0.01), and with the extension of course increased significantly; body weight decreased significantly (126±8vs.160±12, P<0.01); insulin levels increased significantly at first weeks (8.51±4.95vs.1.25±1.33, P <0.01), but in the third weeks the insulin levels decreased significantly compared to the control group (2.99±0.86vs.3.75±1.34, P <0.01), with the extension of course insulin levels drop significantly.Histopathological observation:pancreatic islet in control group were round or oval with a complete clear boundaries; the number of pancreatic islet in model group were significantly reduced, pancreatic atrophy, smaller volume, islet unclear boundaries, islet cell degeneration, edema, nuclear atrophy. The control group glomerular structure was clear, no proliferation of mesangial matrix and basement membrane thickening, interstitial inflammation and fibrosis. The model group increased glomerular volume; glomerular cysts reduced; glomerular epithelial cells arranged in disorder, swelling; glomerular capillary lumen narrowing, partial occlusion, consistent with diabetic nephropathy in other animal.2. Bone marrow mesenchymal stem cells were cultured, identificated and labeled in vitro:the3rd generation of bone marrow mesenchymal stem cells were spindle-shaped whirlpool. Flow cytometry surface marker identification of CD90(95.3%), CD105(99.4%) were positive, CD31(0%), CD34(0%) were negative, according to bone marrow mesenchymal stem cell growth characteristics. and the DAPI labeled was successful,the label rate≥98%.3. Detection of blood glucose, blood lipid and renal function in tree shrews after transplantation,:The polydipsia, polyuria, weight loss of tree shrew in DN+MSC group were improved compared to the DN group, but no significant change of body weight (128±8vs.122±11, P>0.05); blood glucose after treatment21d decreased significantly (13.98±4.16vs.21.89±3.87, P<0.01); the treatment at21d DN+MSC triglyceride (0.99±0.15vs.1.34±0.29, P<0.01), total cholesterol (4.32±0.21vs.6.38±0.49, P<0.01),24h urine volume (12.74±1.82vs.20.67±1.43, P<0.01) was significantly lower than in the group with DN, but still higher than that of control group (P<0.01); urea (12.65±1.30vs.16.16±2.55, P＜0.05),24h urine protein (4.09±0.51vs.5.08±0.29, P<0.05), insulin (0.78±0.24vs.0.26±0.14, P<0.05), creatinine (35.06±4.13vs.44.17±5.05, P＜0.05) level was not obviously decreased compared to the DN group, and increased with the extension of course.Implantation of BMSCs after21d pancreas of DN group islet damage no significant difference than the DN group; kidney glomerular hypertrophy,glomerular capillary constriction,renal cysts narrow more ease than the DN group.4.21days after injected with DAPI labeled BMSC twice, the pancreas, kidney were made into frozen sections, observed under fluorescent microscope, visible around the islets and renal interstitial with a small amount of blue fluorescent substance. It indicated that stem cell can reach the kidney and survival.Conclusion:1. This experiment adopts the high sugar-fat feed add four joint STZ (100mg/kg) on an empty stomach abdominal cavity injection method, a preliminary established tree shrews model creation and evaluation of diabetic nephropathy.2. Tissue adherent culture method is used to obtain cultivation tree shrews between bone marrow mesenchymal stem cells, in training through3generations, CD90, CD105and CD31, CD34as tree shrews between bone marrow mesenchymal stem cell antigen expression of appraisal, CD90, CD105were95.3%,99.4%, respectively, with people or other animals BMSCs analysis results are consistent.3. The success of the experiment using DAPI tag tree shrews between bone marrow mesenchymal stem cells, mark rate≥98%.4. This experiment of cell labeling by DAPI technology is established. DAPI labelled BMSCs transplantation after21d,DN+MSC group of tree shrews level of blood sugar, blood lipid, renal function, is a significant reduction in the DN group and before treatment, the insulin levels in the DN group and before treatment was increased, that BMSCs can significantly lower the blood sugar, blood lipid content in DN tree shrews, improve renal function.5. DAPI labelled BMSCs transplantation after21d, pancreatic tissue for forzen section, fluorescence microscope with a small amount of DAPI labeled cells distributed in tree shrews islet around, but the pancreatic pathology structure compared with DN group was no significant difference, the therapeutic effect of BMSCs in tree shrews diabetes also exist of pancreas damage repair mechanism.6. DAPI labelled BMSCs transplantation after21d, kidney tissue for forzen section, fluorescence microscope with DAPI labeled cells in renal interstitium, pathological tissue slice observation found glomerular hypertrophy, glomerular capillary narrowing, bowman’s capsule cavity to reduce the reduce in the DN group; Prompt BMSCs may be involved in tree shrews diabetic nephropathy renal injury repair, thus changing the structure and function. |
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