【摘要】：目的:蛋白激酶Cα(PKCα)參與尿濃縮功能的調(diào)節(jié),但具體機制尚不清楚。本實驗研究PKCα對水通道蛋白2(aquaporin 2,AQP2)轉(zhuǎn)運和細胞骨架(cytoskeleton,CSK)的分布的作用。 方法:構(gòu)建穩(wěn)定轉(zhuǎn)染AQP2-GFP融合蛋白的小鼠內(nèi)髓集合管上皮細胞(mIMCD_3)株,采用免疫印跡技術(shù)檢測轉(zhuǎn)染效率;體外培養(yǎng)AQP2-GFP mIMCD_3細胞,使用脂質(zhì)體2000分別轉(zhuǎn)染PKCαshRNA質(zhì)粒、PKCα顯性激活型質(zhì)粒(PKCαA25E)及PKCαscramble shRNA陰性對照質(zhì)粒,48h后收集部分細胞行免疫印跡檢測轉(zhuǎn)染效率,部分細胞使用去氨加壓素(DdAVP)刺激,分別收集刺激后0分鐘、30分鐘、以及DdAVP刺激30分鐘后洗脫2小時細胞,采用免疫熒光共聚焦技術(shù)檢測AQP2以及微管的分布變化。 結(jié)果:成功構(gòu)建了穩(wěn)定表達AQP2-GFP的mIMCD_3細胞株;使用PKCαshRNA轉(zhuǎn)染AQP2- mIMCD_3細胞后,轉(zhuǎn)染組PKCα表達明顯減少;與PKCα顯性激活組以及空質(zhì)粒轉(zhuǎn)染組相比,PKCαshRNA轉(zhuǎn)染組使用DdAVP處理30分鐘后,AQP2及微管的分布未見明顯改變。 結(jié)論:PKCα參與了AQP2轉(zhuǎn)運過程的調(diào)節(jié),這一調(diào)節(jié)可能通過影響微管的動力學特性及分布來介導。
[Abstract]:Objective: protein kinase C 偽 (PKC 偽) is involved in the regulation of urinary concentration, but the mechanism remains unclear. The effects of PKC 偽 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 偽 shRNA plasmid, PKC 偽 dominant activated plasmid (PKC 偽 A25E) and PKC 偽 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 偽 in the transfected AQP2- mIMCD_3 cells decreased significantly after transfection with PKC 偽 shRNA. Compared with PKC 偽 dominant activation group and empty plasmid transfection group, the distribution of AQP2 and microtubules in PKC 偽 shRNA transfection group did not change significantly after 30 minutes of DdAVP treatment. Conclusion: PKC 偽 is involved in the regulation of AQP2 transport, which may be mediated by affecting the dynamics and distribution of microtubules.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363

【共引文獻】

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本文編號：2317088