转染外源性prohibitin在缺氧性肾小管上皮细胞损伤中的作用及机制

发布时间：2018-12-21 08:27

【摘要】：目的探讨转染外源性抗增殖蛋白(prohibitin, PHB)基因在缺氧性肾小管上皮细胞(renal tubular epithelial cell, RTEC)损伤中的作用及机制,为临床防治肾间质纤维化(renal interstitial fibrosis, RIF)提供新思路。方法构建pcDNA3.1(+)-PHB1贡粒、pcDNA3.1(+)-PHB2质粒及pcDNA3.1(+)-eGFP质粒,利用脂质体Lipofectamine2000转染体外培养肾小管上皮细胞系(NRK-52E),采用表达绿色荧光的阴性对照pcDNA3.1(+)-eGFP在倒置荧光显微镜下检测转染效率。转染48h后随机分为2组：正常组和模型组,正常组NRK-52E细胞置于培养箱继续培养,模型组NRK-52E细胞置于真空干燥罐中,负压吸引器抽尽残余空气,充以含体积分数为95%N2+5%C02的缺氧气体,密封的方法建立缺氧性RTEC损伤模型。模型组和正常组各设立5个亚组：空白对照组、阴性质粒对照组、脂质体对照组、转染PHB1组及转染PHB2组。造模12h复氧1h后扫描电镜观察NRK-52E细胞形态,实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction, RT-PCR)法检测NRK-52E细胞PHB1、PHB2及α-平滑肌肌动蛋白(a-smooth muscel actin, α-SMA) mRNA表达,免疫印迹(Western blot)法检测NRK-52E细胞PHB1、PHB2及a-SMA蛋白表达,流式细胞仪检测NRK-52E细胞线粒体膜电位,比色法检测NRK-52E细胞培养上清液丙二醛(malondialdehyde, MDA)含量。结果1.成功复苏NRK-52E细胞后按3×105的个数接种到6孔细胞培养板,24h后转染时细胞可达到80-90%的汇合度。按照脂质体Lipofectamine2000说明书推荐的比例转染NRK-52E细胞,转染效率约为80%。 2.(1)正常组和模型组中转染PHB1组、转染PHB2组的NRK-52E细胞PHB1、PHB2的mRNA和蛋白表达量均显著高于相应各组中空白对照组、阴性对照组、脂质体对照组(P0.05),而空白对照组、阴性对照组、脂质体对照组之间无显著性差异(P0.05)；(2)正常组和模型组中转染PHB1组、转染PHB2组的NRK-52E细胞α-SMA mRNA和蛋白表达量均显著低于相应各组中空白对照组、阴性对照组、脂质体对照组(P0.05),而空白对照组、阴性对照组、脂质体对照组之间无显著性差异(P0.05)；(3)与正常组各亚组比较,模型组中相应各亚组NRK-52E细胞PHB1、PHB2mRNA和蛋白表达量均显著降低(P0.05),而α-SMA mRNA表达量均显著增高(P0.05) 3.(1)正常组和模型组中的转染PHB1组、转染PHB2组NRK-52E细胞的线粒体膜电位均显著高于相应各组中空白对照组、阴性对照组、脂质体对照组(P0.05),而空白对照组、阴性对照组、脂质体对照组之间无显著性差异(P0.05)；(2)与正常组各亚组比较,模型组中相应各亚组NRK-52E细胞线粒体膜电位均显著降低(P0.05)。 4.(1)正常组和模型组中转染PHB1组、转染PHB2组NRK-52E细胞的MDA含量均显著低于相应各组中空白对照组、阴性对照组、脂质体对照组(P0.05),而空白对照组、阴性对照组、脂质体对照组之间无显著性差异(P0.05)；(2)与正常组各亚组比较,模型组中相应各亚组NRK-52E细胞MDA含量均显著升高(P0.05)。结论转染外源性PHB1和PHB2基因表达上调后,可能通过增加RTEC线粒体膜电位稳定性、降低MDA的产生及α-SMA的表达量,而起到减轻缺氧性RTEC损伤的作用。
[Abstract]:Objective To study the role and mechanism of exogenous anti-proliferative protein (PHB) gene in the injury of renal tubular epithelial cell (RTEC), and to provide a new thought for clinical prevention and treatment of renal interstitial fibrosis (RIF). Methods The pcDNA3. 1 (+)-PHB1 tribute, the pcDNA3.1 (+)-PH2 plasmid and the pcDNA3.1 (+)-eGFP plasmid were constructed, and the tubular epithelial cell line (NRK-52E) was cultured in vitro by lipofectamine 2000. The negative control pcDNA3.1 (+)-eGFP expressing the green fluorescence was used to detect the transfection efficiency under the inverted fluorescence microscope. Rate. After transfection for 48 h, the cells were randomly divided into 2 groups: normal group and model group, normal group NRK-52E cells were placed in the incubator to continue to be cultured, the NRK-52E cells in the model group were placed in the vacuum drying tank, the negative pressure suction device was used to draw the residual air, and the oxygen-deficient gas with the volume fraction of 95% N2 + 5% C02 was filled. Method for establishing oxygen-deficient RTEC damage model by body and sealing method The model group and the normal group were divided into 5 subgroups: the blank control group, the negative plasmid control group, the liposome control group, the transfection of the PHB1 group and the transfection of the PHB2. The expression of NRK-52E cells, PHB1, PHB2 and human-smooth muscle actin (a-smooth muscel actin, antigen-SMA) mRNA and immunoblotting (Western blot) were used to detect the PHB-52E cells PHB-52E. 1. The expression of PH2 and a-SMA protein, the mitochondrial membrane potential of NRK-52E cells was detected by flow cytometry, and the content of malondialdehyde (MDA) in the cell culture of NRK-52E was detected by colorimetric method. Quantity. Results 1. After successful resuscitation of NRK-52E cells, the cells were inoculated into 6-hole cell culture plates by the number of 3-105, and the cells can reach 80-90% after 24-hour transfection. Confluence. The NRK-52E cells were transfected with the ratio recommended in the Lipofectamine2000 specification and the transfection efficiency was approximately 80 The expression of PHB1 and PHB2 of NRK-52E cells in the PHB2 group was significantly higher than that in the control group, the negative control group and the liposome control group (P0.05). In the negative control group, there was no significant difference between the control group and the control group (P0.05); (2) in the normal group and the model group, the PHB1 group was transfected, and the amount of the NRK-52E cells transfected with the PHB2 group was significantly lower than that in the blank control group, the negative control group and the liposome control group (P There was no significant difference between the control group, the negative control group and the liposome control group (P0.05). (3) There was no significant difference in the expression of PHB1, PHB2mRNA and protein in the corresponding subgroups in the model group (P <0.05). 0. 05), and the amount of SMA-SMA mRNA was significantly higher (P <0.05). P0. 05) 3. (1) The mitochondrial membrane potential of NRK-52E cells in the normal and model groups was significantly higher than that in the control group, the negative control group and the liposome control group (P0.05). There was no significant difference between the group, the negative control group and the liposome control group (P0.05). (2) The mitochondrial membrane potential of the corresponding sub-group NRK-52E in the model group was significantly reduced in the model group compared with each subgroup in the normal group. (P (0. 05). 4. (1) The content of MDA in the normal group and the model group was significantly lower than that of the control group, the negative control group and the liposome control group (P0.05) in the control group, the negative control group and the liposome control group. (2) The MDA content of NRK-52E cells in the corresponding subgroups was significant in the model group compared with each subgroup in the normal group. Conclusion The increase of the expression of exogenous PHB1 and PHB2 gene can decrease the stability of the mitochondrial membrane potential of the RTEC, decrease the production of MDA and the expression of the antigen-SMA, and play a role in reducing the deficiency.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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