【摘要】：目的：通過小分子干擾RNA(siRNA)技術(shù)，建立大鼠關(guān)節(jié)軟骨細(xì)胞中酸敏感離子通道1a(ASIC1a)表達(dá)沉默模型，觀察ASIC1a mRNA及其蛋白的相對(duì)表達(dá)量的變化，檢測ASIC1a通道不同表達(dá)水平的Ca2+內(nèi)流變化以及對(duì)酸化誘導(dǎo)細(xì)胞凋亡的影響，并通過監(jiān)測凋亡相關(guān)基因Bax、Bcl-2mRNA的相對(duì)表達(dá)水平，研究酸敏感離子通道1a的表達(dá)沉默對(duì)AA大鼠關(guān)節(jié)軟骨細(xì)胞凋亡可能的作用機(jī)制。 方法：通過化學(xué)合成法合成特異性熒光短鏈ASIC1a siRNA-FAM，，使用Lipofectamine2000轉(zhuǎn)染試劑盒將ASIC1a siRNA轉(zhuǎn)染入關(guān)節(jié)軟骨細(xì)胞，采用熒光顯微鏡、流式細(xì)胞術(shù)、實(shí)時(shí)熒光定量PCR(q-RT-PCR)及Western Blot法檢測siRNA轉(zhuǎn)染效率及其對(duì)ASIC1a mRNA和蛋白表達(dá)的抑制作用。同時(shí)采用胞外酸化的方法建立AA大鼠關(guān)節(jié)軟骨細(xì)胞凋亡體外模型，激光共聚焦技術(shù)檢測[Ca2+]i水平，Annexin-V/PI流式細(xì)胞術(shù)、Hoechst33258染色法、Tunel法檢測各組細(xì)胞凋亡情況，免疫細(xì)胞化學(xué)技術(shù)檢測軟骨細(xì)胞分泌的Ⅱ型膠原含量變化，q-RT-PCR檢測凋亡相關(guān)基因Bax、Bcl-2的mRNA的相對(duì)表達(dá)水平。 結(jié)果：1.實(shí)驗(yàn)設(shè)計(jì)的3個(gè)ASIC1a-siRNA均能成功轉(zhuǎn)染入大鼠關(guān)節(jié)軟骨細(xì)胞，且轉(zhuǎn)染siRNA-3后大鼠關(guān)節(jié)軟骨細(xì)胞中ASIC1a mRNA表達(dá)顯著低于正常組，最大抑制率為84.8%；Western Blot結(jié)果顯示，轉(zhuǎn)染特異性siRNA-3后ASIC1a蛋白表達(dá)明顯低于正常組； 2.將抑制效率最高的siRNA-3瞬時(shí)轉(zhuǎn)染入關(guān)節(jié)軟骨細(xì)胞后，在胞外酸化條件刺激下觀察關(guān)節(jié)軟骨細(xì)胞外Ca2+內(nèi)流情況，發(fā)現(xiàn)沉默組細(xì)胞Ca2+內(nèi)流幅度明顯低于模型組； 3.將抑制效率最高的siRNA-3瞬時(shí)轉(zhuǎn)染入關(guān)節(jié)軟骨細(xì)胞，在胞外酸化條件刺激下觀察各組細(xì)胞凋亡情況，同時(shí)觀察軟骨細(xì)胞分泌的Ⅱ型膠原含量及凋亡相關(guān)基因Bax、Bcl-2mRNA的相對(duì)表達(dá)水平。結(jié)果表明，與模型組相比，siRNA-3轉(zhuǎn)染引起ASIC1a表達(dá)沉默后AA大鼠關(guān)節(jié)軟骨細(xì)胞凋亡明顯減少，Ⅱ型膠原表達(dá)量顯著增多，并且沉默組Bax/Bcl-2比值較模型組明顯下調(diào)。 結(jié)論：1. siRNA轉(zhuǎn)染可誘導(dǎo)大鼠關(guān)節(jié)軟骨細(xì)胞ASIC1a表達(dá)沉默，是研究酸敏感離子通道對(duì)軟骨細(xì)胞功能影響的可靠模型； 2.基因水平干預(yù)ASIC1a表達(dá)可導(dǎo)致軟骨細(xì)胞的細(xì)胞膜鈣通透性降低； 3. siRNA-3轉(zhuǎn)染對(duì)胞外酸化刺激條件下大鼠關(guān)節(jié)軟骨細(xì)胞的凋亡具有保護(hù)作用，其機(jī)制可能與其沉默ASIC1a的表達(dá)從而促進(jìn)Bcl-2表達(dá)、抑制Bax的表達(dá)有關(guān)，提示酸敏感離子通道1a可能在AA大鼠關(guān)節(jié)軟骨細(xì)胞的代謝過程中發(fā)揮著重要作用。
[Abstract]:Aim: to establish a silencing model of acid-sensitive ion channel 1a (ASIC1a) expression in rat articular chondrocytes by small molecular interference (RNA (siRNA) technique, and to observe the changes of the relative expression of ASIC1a mRNA and its protein. The changes of Ca2 influx at different levels of ASIC1a channel expression and the effect of acidification-induced apoptosis were detected, and the relative expression level of apoptosis-related gene Bax,Bcl-2mRNA was monitored. To study the possible mechanism of expression silencing of acid-sensitive ion channel 1a on apoptosis of articular chondrocytes in AA rats. Methods: specific fluorescent short chain ASIC1a siRNA-FAM, was synthesized by chemical synthesis. ASIC1a siRNA was transfected into articular chondrocytes by Lipofectamine2000 transfection kit. Fluorescence microscopy and flow cytometry were used to transfect ASIC1a siRNA into articular chondrocytes. Real-time quantitative PCR (q-RT-PCR) and Western Blot were used to detect the transfection efficiency of siRNA and its inhibitory effect on the expression of ASIC1a mRNA and protein. The apoptosis of articular chondrocytes in vitro was established by extracellular acidification in AA rats. The level of [Ca2] I was detected by confocal laser technique, and apoptosis was detected by Annexin-V/PI flow cytometry, Hoechst33258 staining and Tunel assay. The changes of type 鈪

本文編號(hào)：2389418