| Objective:1.Establishing rabbit model of type1diabetes;2.Culturing and dentifying the skin fibroblasts of rabbit in vitro;3.Using inducing agent in our laboratory induce skin fibroblasts of rabbit into pluripotent stem cells;4.Dentifying pluripotent stem cells;5.Transplanting induced autologous stem cell which labeled with DAPI marker to rabbit of type1diseases,and giving the comprehensive evaluation about the therapeutie effete by many methods.Methods:1.Establishing rabbit model of type1diabetes and experimental group:General level health experimental rabbit(n=35),the animals randomly were divided into two groups according to body mass number;normal control group(n=10):rabbits without any treatment;model group(n=20):rabbits injected by four oxygen pyrimidine.Diabetes model group was divided into two groups.therapy group(n=10):the animals of model group treated with autologous stem cell;model control group(n=10):no treatment.2.Culturing and dentifying the skin fibroblasts:Take rabbit skin tissues,culture the cell by organization stick wall and the conventional culture method,and identify fibroblasts by some methods which were cells form,wave form protein immunofluorescence, electron microscope and flow cytometry(FCM).3.Using natural actisubstances(inducing agent) which made in our laboratory to induce rabbit skin fibroblasts into pluripotent stem cells,and identify the characteristies of stem cell marker gene by many methods which include immunofluorescence,Semi-quantitative PCR,flow cytometry.4.Transplanting autologous stem cell which labeled with DAPI marker to rabbit of type1diseases,and giving the comprehensive evaluation about the therapeutie effete:investigate the changes of blood sugar, plasma insulin,and plasma C peptide of the rabbits in control group, model group and treatment group.Through pathological tissue section,electron microscope and frozen sections,observe structure and function of the pancreas and the distribution of the cells.Result:1.The three rabbits injected by four oxygen pyrimidine were dead, the level of blood sugar reach standard and was stable in the rest of the rabbits,and the success rates of model was90%.2.Fibroblasts identification: Morphological observation:Skin fibroblasts began to stick wall after0.5-lh,l-2d later spindle cells and forming colony of varying sizes were observed,and after3-4d,there were fusion of80%cells which were evenly distributed.Waveform protein immunofluorescence:The cell cytoplasm is brown color.Electron microscope: Fibroblasts are flat with a spindle, small size, shape and swelled less; The nuclei are small, oval, and coloring flat deep; In the cytoplasm there are reticulum organelles not develope such as drough endoplasmic.FCM:Fibroblasts wave form protein positive expression rate was100%,it expressed4.5%in blank control group.3.Fibroblasts induction and identification:Immunofluorescence:Oct-4gene of the induced cells have a positive expression.FCM:positive expressions of SSEA-4,C-Myc,Oct-4and Nanog respectively were5.48%,6.10%,6.09%,4.01%. Semi-quantitative PCR:Oct4and Nanog gene of the induced cell relatively express2.3175,0.4071.4.Giving the comprehensive evaluation about the therapeutie effete,4w after transplantation.Blood sugar:the level of blood sugar of therapy group is significantly lower than the model control group (P<0.05), and the therapy group and model control group were higher than that of normal control group (P<0.05).Plasma insulin and plasma c-peptide:The level of plasmainsulin and plasma c-peptide treatment of therapy group are higher than the model control group(P<0.05),and therapy group and model control group significantly lower than that of normal group (P<0.05). Pathological tissue section:Normal control group:There have been enriched islets in the pancreas,most characterized by round or oval with integrity.Treatment group:we can find the less quantity of islets,incomplete integrity and smaller volume,were located nearby the pancreas conduit. Model control group: the islets show the least quantity,the most irregular and the smallest volume.Electron microscope:The apparent nucleus of islet β-cell was characterized by greater volume,rich cytoplasm and developed rough endoplasmic reticulum with larger amounts of secreted granules inside cytoplasm in normal control group.By contrast,the islet β-cells of treatment group held the oval and irregular nucleus with many granules analogous to the normal. However,model control group represented that the atrophic islet β-cells was characterized by smaller volume and significant reduction or disappearance of secretory granules disappearance.5.Distribution of the cells after transplantation:Blue fluorescence cells is visible in the pancreas of treatment group,and it is not seen in model control group.Conclusion:1.Making the model by four oxygen pyrimidine may get high success rate and low mortality,and may get rabbit model of type1diabetes whose blood sugar continuously stable.2.High purity fibroblasts can be obtained by the organization stick wall and the conventional culture method.3.Fibroblasts cells were successfully marked by DAPI,and the marked cell is99-100%.4.Using inducing agent which made in our laboratory can successfully induce skin fibroblasts of rabbit into pluripotent stem cells.5.4w after transplantation,the marked DAPI cells were found in the pancreas, the cell transplantation can reduce the blood sugar of diabetes rabbit,improve the islet function,and repair the damaged the pancreas. |
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