發(fā)布時間：2018-06-10 03:22

  本文選題：Rab + 大電導(dǎo)鈣激活鉀通道�。� 參考：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:眾所周知,大電導(dǎo)鈣激活鉀通道(BK_(Ca))可以將人體不同組織細(xì)胞內(nèi)的鈣離子信號和細(xì)胞興奮性相聯(lián)系,這種生理功能在調(diào)節(jié)人體的血液流動、尿液排出、免疫應(yīng)答等多種生命活動中具有極其重要的作用。BK_(Ca)通道是人體血管平滑肌細(xì)胞膜上的主要離子通道,在血管舒縮功能調(diào)控中起著至關(guān)重要的作用。BK_(Ca)通道由4個a亞基與具有調(diào)節(jié)功能的b亞基組成,其中b亞單位是BK_(Ca)通道在人體血管平滑肌細(xì)胞上最為重要的輔助亞單位,是通道蛋白執(zhí)行其生理功能的關(guān)鍵性因素之一,如果b亞單位出現(xiàn)生理功能異常,就可能引起多種心血管疾病(如高血壓等)。BK_(Ca)通道蛋白在內(nèi)質(zhì)網(wǎng)合成、高爾基體加工之后,還需經(jīng)過一系列復(fù)雜的轉(zhuǎn)運(yùn)機(jī)制并組裝到質(zhì)膜上,才能發(fā)揮其生理功能。小G蛋白家族成員中的Rab在質(zhì)膜內(nèi)側(cè)及多種胞內(nèi)囊泡結(jié)構(gòu)中存在,具有調(diào)節(jié)細(xì)胞信號轉(zhuǎn)導(dǎo)、囊泡轉(zhuǎn)運(yùn)和離子通道活動等生理功能。結(jié)合既往的研究,提示Rab和BK_(Ca)通道蛋白可能存在某種相互聯(lián)系,但是全面深入的研究甚少,為了更好的研究BK_(Ca)通道蛋白的調(diào)節(jié)機(jī)制,我們選取了Rab5作為研究對象,展開實(shí)驗(yàn),探討小G蛋白Rab5對HEK293細(xì)胞大電導(dǎo)鈣激活鉀(BK_(Ca))通道的影響。方法:根據(jù)以往的研究經(jīng)驗(yàn),我們選用的實(shí)驗(yàn)細(xì)胞為人胚腎293細(xì)胞系。首先將含有人血管平滑肌BKca通道a亞單位(hSlo1)的Flag和GFP雙標(biāo)記表達(dá)質(zhì)粒(Flag-hSlo1-GFP)使用脂質(zhì)體轉(zhuǎn)染法和不同形式的Rab5表達(dá)質(zhì)粒(野生型WT,顯性負(fù)向型DN,持續(xù)激活型CA)轉(zhuǎn)染至培養(yǎng)的HEK293細(xì)胞,使用膜片鉗技術(shù)、Western blotting和流式細(xì)胞術(shù),研究小G蛋白Rab5對BK_(Ca)通道的細(xì)胞宏觀電流、BK_(Ca)通道的膜蛋白在HEK293細(xì)胞膜上表達(dá)水平和BK_(Ca)通道的轉(zhuǎn)運(yùn)蛋白的影響。結(jié)果:實(shí)驗(yàn)結(jié)果表明,表達(dá)在HEK293細(xì)胞上的大電導(dǎo)鈣激活鉀(BK_(Ca))通道和天然BK_(Ca)通道幾乎具有相似的電生理學(xué)特性,膜片鉗實(shí)驗(yàn)表明在全細(xì)胞構(gòu)型下,當(dāng)細(xì)胞膜電位(V_m)達(dá)到~+60mV時,Rab5 WT和Rab5 CA可以分別增加BKca通道宏觀電流密度由49.19±3.76pA/pF增加到68.38±5.33pA/pF(P0.05,n=6)和87.16±3.79pA/pF(P0.05,n=6)。而Rab5DN減少BKca通道宏觀電流密度,由49.19±3.76pA/pF減少到34.68±3.37pA/pF(P0.05,n=6)。Western blotting實(shí)驗(yàn)結(jié)果顯示,Rab5 WT和Rab5 CA增加BK_(Ca)通道蛋白在HEK293細(xì)胞膜上的表達(dá)水平,相對表達(dá)量分別是對照組的1.24±0.08倍(Rab5 WT)和1.42±0.13倍(Rab5CA)(P0.05),而Rab5 DN減少BK_(Ca)通道蛋白在HEK293細(xì)胞膜上的表達(dá)水平,相對表達(dá)量是對照組的0.73±0.11(P0.05),兩者都具有顯著的統(tǒng)計學(xué)意義。流式細(xì)胞術(shù)實(shí)驗(yàn)結(jié)果顯示Rab5 WT和Rab5 CA可以明顯增加Flag~+/GFP~+比值,而Rab5 DN明顯減小Flag~+/GFP~+比值(P0.05或0.01),這表明Rab5 WT和Rab5 CA明顯促進(jìn)了BK_(Ca)通道蛋白向細(xì)胞膜上的轉(zhuǎn)運(yùn)。結(jié)論:Rab5能夠明顯增加表達(dá)在HEK293細(xì)胞上的BK_(Ca)電流及細(xì)胞膜上的表達(dá),并可能促進(jìn)了BK_(Ca)通道向細(xì)胞膜的轉(zhuǎn)運(yùn)過程。
[Abstract]:Objective: it is well known that large conductance calcium-activated potassium channel (BK) can link calcium signals in different tissues and cells to cell excitability, a physiological function that regulates the flow of blood and excretion of urine. The immune response plays an extremely important role in many life activities. BK\ + Ca) channel is the main ion channel in human vascular smooth muscle cell membrane. In the regulation of vasomotor and contraction function, the BKG Ca) channel is composed of four subunits a and b subunits with regulatory function, among which b subunit is the most important auxiliary subunit of BK subunit in human vascular smooth muscle cells. It is one of the key factors for channel protein to perform its physiological function. If the subunit b has abnormal physiological function, it may cause the synthesis of channel proteins in the endoplasmic reticulum (ER) and the processing of Golgi body in various cardiovascular diseases (such as hypertension, etc.). It also needs a series of complex transport mechanisms and assembly to the plasma membrane to play its physiological function. The Rab of small G protein family exists in the medial membrane of plasma membrane and many kinds of intracellular vesicle structures, and has the physiological function of regulating cell signal transduction, vesicle transport and ion channel activity. In combination with previous studies, it is suggested that there may be some interrelation between Rab and BKS Ca-channel proteins, but there are few comprehensive and in-depth studies. In order to better study the regulation mechanism of BKKCa-Ca-channel proteins, we selected Rab5 as the object of study and carried out experiments. To investigate the effect of small G protein Rab5 on the large conductance calcium activated potassium (BK / C) channel in HEK293 cells. Methods: according to the previous research experience, we selected the human embryonic kidney 293 cell line. Flag and GFP double labeled expression plasmids, Flag-hSlo1-GFP1, containing a subunit of human vascular smooth muscle BKCA channel, were first transfected into HEK293 cells by liposome transfection and different forms of Rab5 expression plasmids (wild type WT, dominant negative type DNs, continuous activated CA1). Using patch clamp technique, Western blotting and flow cytometry were used to study the effect of small G protein Rab5 on the expression of membrane protein on the membrane of HEK293 cell membrane and the transport protein of BK-1 Ca2 channel. Results: the experimental results showed that the large conductance calcium-activated potassium (BK) and the natural BK-1 / CaC) channels expressed in HEK293 cells had almost similar electrophysiological characteristics, and patch clamp experiments showed that in the whole cell configuration, the two channels had similar electrophysiological characteristics. Rab5WT and Rab5CA could increase the macroscopic current density of BKCA channel from 49.19 鹵3.76pA / PF to 68.38 鹵5.33pA / P0.05P0.05N ~ (6) and 87.16 鹵3.79pAp / p ~ (0.05p ~ (6) respectively when the cell membrane potential reached ~ 60mV. However, Rab5DN decreased the macroscopic current density of BKca channel from 49.19 鹵3.76pA / PF to 34.68 鹵3.37pFN / P0.05P0.05P0.05P0.05P0.05P0. Western blotting results showed that Rab5WT and Rab5CA increased the expression level of BKSI-CaA channel protein on HEK293 cell membrane. The relative expression was 1.24 鹵0.08 times higher than that of control group (1.24 鹵0.08 times) and 1.42 鹵0.13 times of Rab5CAA (P 0.05), respectively, while Rab5 DN reduced the expression level of BKapphica-channel protein on HEK293 cell membrane. The relative expression level of Rab5DN was 0.73 鹵0.11P0.05in the control group. Both of them had significant statistical significance. The results of flow cytometry showed that Rab5WT and Rab5CA could significantly increase the Flag- / GFP~ ratio, while Rab5DN significantly decreased the Flag- / GFP~ ratio (P0.05 or 0.01), which indicated that Rab5WT and Rab5CA significantly promoted the transport of BKGFP-) channel protein to the cell membrane. ConclusionWowRab5 can obviously increase the BKS Ca) current expressed in HEK293 cells and the expression on the cell membrane, and may promote the transport process of BKS Ca) channel to the cell membrane.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R33

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相關(guān)期刊論文 前1條

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本文編號：2001806