ClC-2氯离子通道在RGC-5细胞凋亡中的作用研究

发布时间：2018-06-19 16:46

本文选题：RGC-5 + ClC-2　；参考：《吉林大学》2014年博士论文

【摘要】：视网膜神经节细胞(retinal ganglion cells, RGCs)的凋亡是视神经损伤、青光眼、缺血性视神经病变等眼科疾病的重要病理特征之一，也是导致视野缺损的主要原因。RGC-5细胞系是目前作为视神经保护体外实验模型的一个重要细胞系。谷氨酸是视网膜的主要兴奋性递质，正常情况下不引起毒性。但在眼压升高、视神经供血不足等导致视网膜缺血缺氧时，谷氨酸会大量释放，对视网膜神经节细胞产生毒性作用。谷氨酸的过度表达是导致RGCs凋亡的重要原因。因此，利用谷氨酸诱导的RGC-5凋亡可以作为研究视网膜神经节细胞凋亡及视神经保护的模型。氯离子是生物体内含量最丰富的阴离子，，通过跨膜转运和离子通道参与机体多种生物功能。电压门控性氯离子通道(voltage-gated chloride channnel, ClC)在哺乳动物细胞中广泛表达，ClC-2氯离子通道作为此家族中的一个亚型，是目前研究较为广泛和明确的一种氯离子通道类型，与细胞增殖、凋亡及细胞周期等多种生理功能的调节有关。关于电压门控性氯通道参与细胞凋亡过程己得到广泛证实。线粒体凋亡通路是细胞凋亡的主要途径之一。眼部组织多种细胞的凋亡都是通过线粒体凋亡通路完成，并在多种眼科疾病中发挥作用。Bcl-2基因家族对细胞凋亡的激活和调控起着关键作用，是RGCs主要的内源性凋亡通路。鉴于此，实验以谷氨酸诱导RGC-5细胞凋亡作为研究对象，应用cDNA转染和RNAi基因沉默方法分别改变细胞内ClC-2的表达，观察细胞凋亡、细胞周期变化情况及Bcl-2及Bax蛋白表达、caspase-3、caspase-9酶活性变化，以探讨ClC-2及Bcl-2/Bax调控的线粒体凋亡通路在此过程中的作用。实验中首先应用RT-PCR的方法证实了RGC-5细胞中存在ClC-2mRNA的表达；然后应用细胞免疫荧光染色的方法证实了ClC-2蛋白在RGC-5细胞中存在表达，并定位于细胞浆，为下一步实验提供了基础和依据。文献报道当谷氨酸刺激浓度为lmM时，并作用于RGC-5细胞24小时的凋亡率大约为20%，是较适宜的研究浓度。因此我们将传代后的细胞贴壁生长24小时后，加入含有lmM谷氨酸的无血清DMEM培养液培养24小时，从而得到RGC-5细胞凋亡的模型。进一步实验以谷氨酸诱导的RGC-5细胞凋亡为研究对象，应用ClC-2cDNA技术成功转染细胞，使细胞内ClC-2的mRNA及蛋白质表达均明显升高。应用MTT法、流式细胞仪检测细胞的凋亡及周期变化情况。MTT法结果显示细胞的存活率明显上升，流式细胞仪检测结果表明凋亡率下降，并且细胞周期G1期的细胞比例减少，进入S期细胞增多。以上结果提示ClC-2的过表达对谷氨酸诱导的RGC-5细胞凋亡具有保护作用。随后实验应用RNAi技术转染谷氨酸诱导的RGC-5细胞，RT-PCR及Westernblot结果证实ClC-2的mRNA及蛋白质表达均被明显抑制，应用MTT法、流式细胞仪检测细胞的凋亡及周期变化情况。MTT法结果显示细胞的存活率明显下降，流式细胞仪检测结果表明凋亡率增加，并且细胞周期G1期的细胞比例明显增多，进入S期细胞显著减少。结果证实了抑制ClC-2的表达可以促进细胞的凋亡。以上两部分实验从正反两方面提示ClC-2氯离子通道对谷氨酸诱导的RGC-5细胞凋亡具有保护作用。为了进一步探讨ClC-2氯离子通道如何抑制谷氨酸诱导的RGC-5细胞凋亡作用，我们进行了分子机制的研究。分别采用cDNA转染技术增加RGC-5细胞内ClC-2的表达及RNAi技术抑制RGC-5细胞内ClC-2的表达，观察Bcl-2、Bax蛋白及caspase-3、caspase-9酶活性在各组细胞中的变化。发现与谷氨酸诱导组相比，谷氨酸诱导+ClC-2cDNA转染组Bax蛋白表达、caspase-3、caspase-9酶活性均显著下降，而Bcl-2蛋白表达显著升高；相反，与谷氨酸诱导组相比，谷氨酸诱导+ClC-2RNAi组，Bax蛋白表达、caspase-3、caspase-9酶活性均显著增高，Bcl-2蛋白表达显著降低；以上实验结果说明了ClC-2氯离子通道的抗凋亡作用是通过内源性线粒体凋亡通路进行，并通过上调Bcl-2蛋白表达及下调Bax蛋白来调控细胞凋亡。本研究探讨了在谷氨酸诱导的RGC-5细胞凋亡过程中，ClC-2氯离子通道及线粒体凋亡通路的作用，发现ClC-2氯离子通道对谷氨酸诱导的RGC-5细胞凋亡具有保护作用，而Bcl-2/Bax调控的线粒体凋亡通路可能参与此过程。我们的实验结果提示ClC-2氯离子通道及Bcl-2/Bax调控的线粒体凋亡通路可能成为视神经保护的一个新靶点，为进一步研究视神经保护提供了理论基础和实验依据。
[Abstract]:The apoptosis of retinal ganglion cells ( RGCs ) is one of the important pathological features of optic nerve injury , glaucoma and ischemic optic neuropathy .

ClC - 2 chloride channel ( ClC ) has been widely used in mammalian cells . ClC - 2 chloride channel is one of the main types of cell apoptosis .

In view of this , the expression of ClC - 2 in the cells was investigated by cDNA transfection and RNAi gene silencing , and the changes of apoptosis , cell cycle and the expression of Bcl - 2 and Bax protein , caspase - 3 and caspase - 9 activities were observed . The effects of ClC - 2 and Bcl - 2 / Bax regulation on the apoptosis of mitochondria were investigated .

In the experiment , the expression of ClC - 2 mRNA in RGC - 5 cells was confirmed by RT - PCR .
Then , the expression of ClC - 2 protein in RGC - 5 cells was confirmed by immunofluorescence staining method .

It was reported that when the concentration of glutamic acid was lmM and the apoptosis rate of RGC - 5 cells for 24 hours was about 20 % , it was suitable to study the concentration . Therefore , after 24 hours of cell wall growth , we cultured the serum - free DMEM culture solution containing lmM glutamic acid for 24 hours , thus obtaining the model of RGC - 5 apoptosis .

The apoptosis and protein expression of ClC - 2 cells were significantly increased by using ClC - 2 cDNA technique . The results of MTT assay showed that the survival rate of cells increased significantly . The results of MTT assay showed that the apoptosis rate of cells decreased and the percentage of cells in G1 phase decreased and the number of cells entering S phase increased . The results suggested that the overexpression of ClC - 2 had protective effect on the apoptosis of RGC - 5 cells induced by glutamate .

The results of RT - PCR and Western blot showed that the expression of ClC - 2 mRNA and protein were significantly inhibited . The results of MTT assay showed that the survival rate of cells decreased significantly . The results of MTT assay showed that the apoptosis rate of cells increased and the percentage of cells in G1 phase increased significantly . The results demonstrated that inhibition of ClC - 2 expression could promote apoptosis of cells .

In order to further investigate the effect of ClC - 2 chloride channel on the apoptosis of RGC - 5 cells induced by glutamate , the expression of ClC - 2 and the expression of ClC - 2 in RGC - 5 cells were investigated by cDNA transfection . The expression of Bcl - 2 , Bax protein and caspase - 3 and caspase - 9 were observed .
In contrast , the expression of Bax protein , caspase - 3 and caspase - 9 were significantly higher than that of Glu - induced group , and the expression of Bcl - 2 protein was significantly decreased .
The results showed that the anti - apoptotic effect of ClC - 2 chloride channel was regulated by endogenous mitochondrial apoptotic pathway , and apoptosis was regulated by up - regulation of Bcl - 2 protein expression and down - regulation of Bax protein .

In this study , the role of ClC - 2 chloride channel and mitochondria apoptosis pathway during the apoptosis of RGC - 5 cells induced by glutamate was investigated . It was found that ClC - 2 chloride channel had protective effect on glutamate - induced apoptosis of RGC - 5 cells , while Bcl - 2 / Bax regulated mitochondrial apoptosis pathway might be involved in this process . Our experimental results suggest that the pathway of mitochondrial apoptosis regulated by ClC - 2 chloride channel and Bcl - 2 / Bax may become a new target of optic nerve protection , which provides theoretical basis and experimental basis for further study of optic nerve protection .
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R77

【参考文献】