本文關(guān)鍵詞： 骨髓間充質(zhì)干細胞 阿霉素腎病 干預(yù)作用 蛋白尿 mTOR p-mTOR nephrin　出處：《福建醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：1、大鼠骨髓間充質(zhì)干細胞（bone marrow mesenchymal stem cells，BMSCs）的分離及培養(yǎng)，并鑒定其相關(guān)生物學(xué)特性； 2、建立阿霉素腎病大鼠模型； 3、檢測生化指標，電鏡觀察，應(yīng)用普通PCR和Western blot技術(shù)觀察骨髓間充質(zhì)干細胞對阿霉素腎病大鼠的干預(yù)作用及mTOR的表達。 方法：1、提取、培養(yǎng)與鑒定SD大鼠骨髓間充質(zhì)干細胞---通過大鼠淋巴細胞分離液得到單個核細胞層，再通過密度梯度離心法和貼壁篩選法，不斷傳代、擴增，并通過流式細胞儀鑒定表面標志物質(zhì)、同時運用成骨及成脂誘導(dǎo)方法鑒定骨髓間充質(zhì)干細胞生物學(xué)特性； 2、大鼠阿霉素腎病模型建立---非麻醉狀態(tài)下尾靜脈一次性按7.5mg/Kg體重注射鹽酸阿霉素，14天后，綜合各項結(jié)果：一般表現(xiàn)，24h尿蛋白定量、肌酐等各項生化指標，結(jié)合腎臟組織HE染色及電鏡觀察大鼠阿霉素腎病模型； 3、骨髓間充質(zhì)干細胞對阿霉素腎病大鼠的干預(yù)作用及mTOR的表達---實驗分組：模型組；MSC組；正常對照組；應(yīng)用普通PCR和western blot技術(shù)動態(tài)觀察足細胞裂孔膜蛋白nephrin及哺乳動物雷帕霉素靶蛋白mTOR不同時間點mRNA水平和蛋白水平的表達情況。 結(jié)果：1、大鼠BMSCs細胞易于分離、培養(yǎng)，而且具有間質(zhì)細胞的表面抗原特征，純度高，活性高及多向分化潛能； 2、注射鹽酸阿霉素14天后，綜合各項結(jié)果判定本方法建立大鼠阿霉素腎病模型成功，可以實驗； 3、干預(yù)后各時間點，PCR檢測模型組nephrinmRNA表達量均下降，隨時間推移下降越明顯，干細胞干預(yù)后nephrinmRNA表達量出現(xiàn)上升（較模型組），但相對于正常組仍然下降，Western blot檢測nephrin同上。干預(yù)后1周及3周，Western blot表明模型組mTOR條帶稍微增粗，p-mTOR條帶模型組也增粗，且經(jīng)干細胞干預(yù)后條帶變窄，但相對正常組仍然變寬�；叶戎党霈F(xiàn)相應(yīng)變化，干預(yù)后2w，Western blot表明模型組mTOR條帶稍微增粗，p-mTOR條帶模型組增粗較明顯，且經(jīng)干細胞干預(yù)后條帶變窄窄，但相對正常組仍然變寬�；叶戎党霈F(xiàn)相應(yīng)變化. 結(jié)論：1、可以通過密度梯度離心法結(jié)合貼壁篩選法，成功地從大鼠骨髓中分離出高純度、高活性和具有多向分化潛能的大鼠骨髓間充質(zhì)干細胞； 2、可以經(jīng)尾靜脈一次性按7.5mg/Kg體重注射鹽酸阿霉素，建立大鼠阿霉素腎病模型，時間為14天； 3、MSC保護阿霉素腎病大鼠的可能機制是：MSC的輸注抑制了腎組織mTOR信號通路的激活，，通過維持足細胞裂孔膜蛋白nephrin的穩(wěn)定進而維持足細胞裂孔膜結(jié)構(gòu)的完整，改善了蛋白尿，保護了因阿霉素導(dǎo)致的腎小球足細胞的損害，達到了延緩疾病進展的目的。
[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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R692


本文編號：1499574