蛋白激酶C-α对水通道蛋白2转运的调节作用研究
发布时间：2018-11-07 17:39
【摘要】：目的:蛋白激酶Cα(PKCα)参与尿浓缩功能的调节,但具体机制尚不清楚。本实验研究PKCα对水通道蛋白2(aquaporin 2,AQP2)转运和细胞骨架(cytoskeleton,CSK)的分布的作用。 方法:构建稳定转染AQP2-GFP融合蛋白的小鼠内髓集合管上皮细胞(mIMCD_3)株,采用免疫印迹技术检测转染效率;体外培养AQP2-GFP mIMCD_3细胞,使用脂质体2000分别转染PKCαshRNA质粒、PKCα显性激活型质粒(PKCαA25E)及PKCαscramble shRNA阴性对照质粒,48h后收集部分细胞行免疫印迹检测转染效率,部分细胞使用去氨加压素(DdAVP)刺激,分别收集刺激后0分钟、30分钟、以及DdAVP刺激30分钟后洗脱2小时细胞,采用免疫荧光共聚焦技术检测AQP2以及微管的分布变化。 结果:成功构建了稳定表达AQP2-GFP的mIMCD_3细胞株;使用PKCαshRNA转染AQP2- mIMCD_3细胞后,转染组PKCα表达明显减少;与PKCα显性激活组以及空质粒转染组相比,PKCαshRNA转染组使用DdAVP处理30分钟后,AQP2及微管的分布未见明显改变。 结论:PKCα参与了AQP2转运过程的调节,这一调节可能通过影响微管的动力学特性及分布来介导。
[Abstract]:Objective: protein kinase C 伪 (PKC 伪) is involved in the regulation of urinary concentration, but the mechanism remains unclear. The effects of PKC 伪 on the transport of aquaporin-2 (aquaporin _ 2) and the distribution of cytoskeleton (cytoskeleton,CSK) were studied. Methods: murine intramedullary collecting duct epithelial cells (mIMCD_3) were stably transfected with AQP2-GFP fusion protein and the transfection efficiency was detected by Western blot. AQP2-GFP mIMCD_3 cells were cultured in vitro. PKC 伪 shRNA plasmid, PKC 伪 dominant activated plasmid (PKC 伪 A25E) and PKC 伪 scramble shRNA negative control plasmid were transfected with liposome 2000, respectively. Some cells were stimulated with desmotensin (DdAVP). The cells were washed out at 0 min, 30 min after stimulation and 30 min after stimulation by DdAVP. The distribution of AQP2 and microtubules were detected by immunofluorescence confocal technique. Results: the mIMCD_3 cell lines expressing AQP2-GFP stably were successfully constructed, and the expression of PKC 伪 in the transfected AQP2- mIMCD_3 cells decreased significantly after transfection with PKC 伪 shRNA. Compared with PKC 伪 dominant activation group and empty plasmid transfection group, the distribution of AQP2 and microtubules in PKC 伪 shRNA transfection group did not change significantly after 30 minutes of DdAVP treatment. Conclusion: PKC 伪 is involved in the regulation of AQP2 transport, which may be mediated by affecting the dynamics and distribution of microtubules.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
本文编号：2317088
[Abstract]:Objective: protein kinase C 伪 (PKC 伪) is involved in the regulation of urinary concentration, but the mechanism remains unclear. The effects of PKC 伪 on the transport of aquaporin-2 (aquaporin _ 2) and the distribution of cytoskeleton (cytoskeleton,CSK) were studied. Methods: murine intramedullary collecting duct epithelial cells (mIMCD_3) were stably transfected with AQP2-GFP fusion protein and the transfection efficiency was detected by Western blot. AQP2-GFP mIMCD_3 cells were cultured in vitro. PKC 伪 shRNA plasmid, PKC 伪 dominant activated plasmid (PKC 伪 A25E) and PKC 伪 scramble shRNA negative control plasmid were transfected with liposome 2000, respectively. Some cells were stimulated with desmotensin (DdAVP). The cells were washed out at 0 min, 30 min after stimulation and 30 min after stimulation by DdAVP. The distribution of AQP2 and microtubules were detected by immunofluorescence confocal technique. Results: the mIMCD_3 cell lines expressing AQP2-GFP stably were successfully constructed, and the expression of PKC 伪 in the transfected AQP2- mIMCD_3 cells decreased significantly after transfection with PKC 伪 shRNA. Compared with PKC 伪 dominant activation group and empty plasmid transfection group, the distribution of AQP2 and microtubules in PKC 伪 shRNA transfection group did not change significantly after 30 minutes of DdAVP treatment. Conclusion: PKC 伪 is involved in the regulation of AQP2 transport, which may be mediated by affecting the dynamics and distribution of microtubules.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
【共引文献】
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2 闻颖;TNFα引起肝肾综合征肾小球滤过率下降的机制研究[D];中国医科大学;2007年
相关硕士学位论文 前3条
1 王桃霞;蛋白激酶C-α对内髓集合管上皮细胞系mIMCD3骨架actin的影响[D];华中科技大学;2010年
2 路红;AQP1在Wistar大鼠肾脏发育过程中表达的研究[D];吉林大学;2009年
3 何静妮;内毒素致肾髓质上皮细胞损伤与水通道蛋白2表达的关系[D];中国医科大学;2009年
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