本文關(guān)鍵詞： 小G蛋白家族 大電導(dǎo)鈣激活鉀通道 蛋白轉(zhuǎn)運(yùn) 基因轉(zhuǎn)染　出處：《山東醫(yī)藥》2017年08期 　論文類型：期刊論文

【摘要】：目的探討小G蛋白R(shí)ab5對(duì)HEK293細(xì)胞大電導(dǎo)鈣激活鉀通道(BKCa)的影響。方法將對(duì)數(shù)生長(zhǎng)期HEK293細(xì)胞,隨機(jī)分為Control組、Rab5WT組、Rab5CA組和Rab5DN組。采用脂質(zhì)體轉(zhuǎn)染法進(jìn)行轉(zhuǎn)染,Control組轉(zhuǎn)染Flag-h Slo1-GFP質(zhì)粒和對(duì)照質(zhì)粒pc DNA3.1,Rab5WT組轉(zhuǎn)染Flag-h Slo1-GFP質(zhì)粒和Rab5WT質(zhì)粒,Rab5CA組轉(zhuǎn)染Flag-h Slo1-GFP質(zhì)粒和Rab5CA質(zhì)粒,Rab5DN組轉(zhuǎn)染Flag-h Slo1-GFP質(zhì)粒和Rab5DN質(zhì)粒,每組兩種質(zhì)粒總量為4μg,按質(zhì)量比1∶1共轉(zhuǎn)染至3.5 mm培養(yǎng)皿中。轉(zhuǎn)染72 h、熒光顯微鏡下觀察轉(zhuǎn)染效率在70%以上時(shí),采用膜片鉗、Western blotting法和流式細(xì)胞術(shù)觀察Rab5對(duì)HEK293細(xì)胞BKCa的作用。結(jié)果與Control組比較,Rab5WT組、Rab5CA組BKCa全細(xì)胞電流密度明顯增加,而Rab5DN組明顯降低;Rab5WT組、Rab5CA組BKCa膜蛋白的相對(duì)表達(dá)量明顯升高,而Rab5DN組明顯降低(P均0.05);Rab5WT組、Rab5CA組Flag+/GFP+明顯增加,而Rab5DN組明顯降低(P均0.05)。結(jié)論 Rab5能夠明顯增加表達(dá)在HEK293細(xì)胞上的BKCa電流及細(xì)胞膜上的表達(dá),并可促進(jìn)BKCa向質(zhì)膜的轉(zhuǎn)運(yùn)過(guò)程。
[Abstract]:Objective to investigate the effect of small G protein Rab5 on large conductance calcium activated potassium channel (BK Ca) in HEK293 cells. Methods HEK293 cells in logarithmic phase were randomly divided into Control group. Rab5WT group, Rab5CA group and Rab5DN group were transfected by liposome transfection. Flag-h Slo1-GFP plasmid and control plasmid PC DNA3.1 were transfected into Control group. Flag-h Slo1-GFP plasmid and Rab5WT plasmid were transfected into Rab5WT group. Flag-h Slo1-GFP plasmid and Rab5CA plasmid were transfected into Rab5CA group. Flag-h Slo1-GFP plasmid and Rab5DN plasmid were transfected into Rab5DN group, and the total amount of two kinds of plasmids in each group was 4 渭 g. When the transfection efficiency was more than 70% under fluorescence microscope for 72 h, the patch clamp was used when the transfection was carried out in 3.5mm petri dish at the mass ratio of 1: 1. The effect of Rab5 on BKCa of HEK293 cells was observed by Western blotting and flow cytometry. The whole cell current density of BKCa increased significantly in Rab5CA group, but decreased in Rab5DN group. The relative expression of BKCa membrane protein in Rab5WT group was significantly increased, while that in Rab5DN group was significantly lower than that in Rab5DN group. In Rab5WT group, Flag / GFP increased significantly in Rab5CA group. Conclusion Rab5 can significantly increase the expression of BKCa current and cell membrane in HEK293 cells. It can promote the transport of BKCa to plasma membrane.
【作者單位】： 西南醫(yī)科大學(xué)附屬醫(yī)院;西南醫(yī)科大學(xué)心血管醫(yī)學(xué)研究所;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(31300948)
【分類號(hào)】：R329.2
【正文快照】： 大電導(dǎo)鈣激活鉀通道(BKCa)是血管平滑肌細(xì)胞膜上重要的離子通道,可攜帶70%~80%的外向電流,在調(diào)節(jié)血管張力和血壓中發(fā)揮重要作用[1]。我們前期研究發(fā)現(xiàn),BKCa是眾多舒血管藥物的作用靶點(diǎn),其功能活動(dòng)異常與高血壓等密切相關(guān)[2,3]。BKCa蛋白在內(nèi)質(zhì)網(wǎng)合成、高爾基體加工后,需經(jīng)過(guò)一

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