骨髓间充质干细胞对阿霉素肾病大鼠的干预作用及mTOR的表达

发布时间：2018-02-10 03:42

  本文关键词： 骨髓间充质干细胞 阿霉素肾病 干预作用 蛋白尿 mTOR p-mTOR nephrin　出处：《福建医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：1、大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）的分离及培养，并鉴定其相关生物学特性； 2、建立阿霉素肾病大鼠模型； 3、检测生化指标，电镜观察，应用普通PCR和Western blot技术观察骨髓间充质干细胞对阿霉素肾病大鼠的干预作用及mTOR的表达。 方法：1、提取、培养与鉴定SD大鼠骨髓间充质干细胞---通过大鼠淋巴细胞分离液得到单个核细胞层，再通过密度梯度离心法和贴壁筛选法，不断传代、扩增，并通过流式细胞仪鉴定表面标志物质、同时运用成骨及成脂诱导方法鉴定骨髓间充质干细胞生物学特性； 2、大鼠阿霉素肾病模型建立---非麻醉状态下尾静脉一次性按7.5mg/Kg体重注射盐酸阿霉素，14天后，综合各项结果：一般表现，24h尿蛋白定量、肌酐等各项生化指标，结合肾脏组织HE染色及电镜观察大鼠阿霉素肾病模型； 3、骨髓间充质干细胞对阿霉素肾病大鼠的干预作用及mTOR的表达---实验分组：模型组；MSC组；正常对照组；应用普通PCR和western blot技术动态观察足细胞裂孔膜蛋白nephrin及哺乳动物雷帕霉素靶蛋白mTOR不同时间点mRNA水平和蛋白水平的表达情况。 结果：1、大鼠BMSCs细胞易于分离、培养，而且具有间质细胞的表面抗原特征，纯度高，活性高及多向分化潜能； 2、注射盐酸阿霉素14天后，综合各项结果判定本方法建立大鼠阿霉素肾病模型成功，可以实验； 3、干预后各时间点，PCR检测模型组nephrinmRNA表达量均下降，随时间推移下降越明显，干细胞干预后nephrinmRNA表达量出现上升（较模型组），但相对于正常组仍然下降，Western blot检测nephrin同上。干预后1周及3周，Western blot表明模型组mTOR条带稍微增粗，p-mTOR条带模型组也增粗，且经干细胞干预后条带变窄，但相对正常组仍然变宽。灰度值出现相应变化，干预后2w，Western blot表明模型组mTOR条带稍微增粗，p-mTOR条带模型组增粗较明显，且经干细胞干预后条带变窄窄，但相对正常组仍然变宽。灰度值出现相应变化. 结论：1、可以通过密度梯度离心法结合贴壁筛选法，成功地从大鼠骨髓中分离出高纯度、高活性和具有多向分化潜能的大鼠骨髓间充质干细胞； 2、可以经尾静脉一次性按7.5mg/Kg体重注射盐酸阿霉素，建立大鼠阿霉素肾病模型，时间为14天； 3、MSC保护阿霉素肾病大鼠的可能机制是：MSC的输注抑制了肾组织mTOR信号通路的激活，，通过维持足细胞裂孔膜蛋白nephrin的稳定进而维持足细胞裂孔膜结构的完整，改善了蛋白尿，保护了因阿霉素导致的肾小球足细胞的损害，达到了延缓疾病进展的目的。
[Abstract]:Objective to isolate and culture bone marrow mesenchymal stem cells BMSCs from rat bone marrow mesenchymal stem cells (BMSCs) and identify its biological characteristics. 2. Establish adriamycin nephropathy rat model; 3. Biochemical indexes and electron microscope were observed. The intervention effect of bone marrow mesenchymal stem cells on adriamycin nephropathy rats and the expression of mTOR were observed by ordinary PCR and Western blot techniques. Methods the bone marrow mesenchymal stem cells (BMSCs) of SD rats were extracted, cultured and identified. The mononuclear cells were isolated from rat lymphocytes. The mononuclear cells were subcultured and amplified by density gradient centrifugation and adherent screening. The surface markers were identified by flow cytometry and the biological characteristics of bone marrow mesenchymal stem cells were identified by osteogenesis and lipogenesis. 2. The rat adriamycin nephropathy model was established. The caudal vein was injected with adriamycin hydrochloride at a body weight of 7.5 mg / kg for 14 days. The results were as follows: 24 h urinary protein quantification, creatinine and other biochemical indexes. The model of adriamycin nephropathy in rats was observed by HE staining and electron microscope. 3. The intervention effect of bone marrow mesenchymal stem cells on adriamycin nephropathy rats and the expression of mTOR. The expression of mRNA and protein at different time points in podocyte lobes membrane protein (nephrin) and mammalian rapamycin target protein (mTOR) were dynamically observed by PCR and western blot techniques. Results the rat BMSCs cells were easy to be isolated and cultured, and had the characteristics of surface antigen, high purity, high activity and multidirectional differentiation potential of interstitial cells. (2) after 14 days of injection of adriamycin hydrochloride, it was determined that the model of adriamycin nephropathy in rats was successfully established by this method. 3. After intervention, the expression of nephrinmRNA in the model group decreased at each time point after intervention, and the decrease was more obvious with the passage of time. The expression of nephrinmRNA increased after the intervention of stem cells (compared with the model group, but still decreased compared with the normal group. Western blot showed that the mTOR band of the model group was slightly thicker and the p-mTOR band was also thicker than that of the model group at 1 and 3 weeks after the intervention. After the intervention of stem cells, the band became narrower, but still wider than that of the normal group, and the gray value changed accordingly. After 2 weeks of intervention, the mTOR bands in the model group were slightly thickened and p-mTOR bands were slightly thickened, and the bands were narrower and narrower after the intervention of stem cells. However, the relative normal group is still wider and the gray value changes accordingly. Conclusion the bone marrow mesenchymal stem cells with high purity, high activity and multidirectional differentiation potential can be successfully isolated from rat bone marrow by density gradient centrifugation and adherent screening. (2) adriamycin hydrochloride could be injected into the tail vein at a single dose of 7.5 mg / kg to establish a rat model of adriamycin nephropathy for 14 days. 3The possible mechanism of protecting adriamycin-induced nephropathy in rats with doxorubicin nephropathy was that the infusion of mTOR inhibited the activation of mTOR signaling pathway in renal tissue, and improved proteinuria by maintaining the stability of podocyte membrane protein (nephrin) and thus maintaining the integrity of podocyte membrane structure in rats with adriamycin nephropathy. It protects the damage of glomerular podocytes caused by adriamycin and delays the progress of the disease.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692


本文编号：1499574