酸敏感离子通道1a表达沉默对AA大鼠关节软骨细胞凋亡的影响
发布时间：2018-12-21 20:29
【摘要】：目的：通过小分子干扰RNA(siRNA)技术，建立大鼠关节软骨细胞中酸敏感离子通道1a(ASIC1a)表达沉默模型，观察ASIC1a mRNA及其蛋白的相对表达量的变化，检测ASIC1a通道不同表达水平的Ca2+内流变化以及对酸化诱导细胞凋亡的影响，并通过监测凋亡相关基因Bax、Bcl-2mRNA的相对表达水平，研究酸敏感离子通道1a的表达沉默对AA大鼠关节软骨细胞凋亡可能的作用机制。 方法：通过化学合成法合成特异性荧光短链ASIC1a siRNA-FAM，，使用Lipofectamine2000转染试剂盒将ASIC1a siRNA转染入关节软骨细胞，采用荧光显微镜、流式细胞术、实时荧光定量PCR(q-RT-PCR)及Western Blot法检测siRNA转染效率及其对ASIC1a mRNA和蛋白表达的抑制作用。同时采用胞外酸化的方法建立AA大鼠关节软骨细胞凋亡体外模型，激光共聚焦技术检测[Ca2+]i水平，Annexin-V/PI流式细胞术、Hoechst33258染色法、Tunel法检测各组细胞凋亡情况，免疫细胞化学技术检测软骨细胞分泌的Ⅱ型胶原含量变化，q-RT-PCR检测凋亡相关基因Bax、Bcl-2的mRNA的相对表达水平。 结果：1.实验设计的3个ASIC1a-siRNA均能成功转染入大鼠关节软骨细胞，且转染siRNA-3后大鼠关节软骨细胞中ASIC1a mRNA表达显著低于正常组，最大抑制率为84.8%；Western Blot结果显示，转染特异性siRNA-3后ASIC1a蛋白表达明显低于正常组； 2.将抑制效率最高的siRNA-3瞬时转染入关节软骨细胞后，在胞外酸化条件刺激下观察关节软骨细胞外Ca2+内流情况，发现沉默组细胞Ca2+内流幅度明显低于模型组； 3.将抑制效率最高的siRNA-3瞬时转染入关节软骨细胞，在胞外酸化条件刺激下观察各组细胞凋亡情况，同时观察软骨细胞分泌的Ⅱ型胶原含量及凋亡相关基因Bax、Bcl-2mRNA的相对表达水平。结果表明，与模型组相比，siRNA-3转染引起ASIC1a表达沉默后AA大鼠关节软骨细胞凋亡明显减少，Ⅱ型胶原表达量显著增多，并且沉默组Bax/Bcl-2比值较模型组明显下调。 结论：1. siRNA转染可诱导大鼠关节软骨细胞ASIC1a表达沉默，是研究酸敏感离子通道对软骨细胞功能影响的可靠模型； 2.基因水平干预ASIC1a表达可导致软骨细胞的细胞膜钙通透性降低； 3. siRNA-3转染对胞外酸化刺激条件下大鼠关节软骨细胞的凋亡具有保护作用，其机制可能与其沉默ASIC1a的表达从而促进Bcl-2表达、抑制Bax的表达有关，提示酸敏感离子通道1a可能在AA大鼠关节软骨细胞的代谢过程中发挥着重要作用。
[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
本文编号：2389418
[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
本文编号：2389418
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