本文選題：RGC-5 + ClC-2　； 參考：《吉林大學(xué)》2014年博士論文

【摘要】：視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(retinal ganglion cells, RGCs)的凋亡是視神經(jīng)損傷、青光眼、缺血性視神經(jīng)病變等眼科疾病的重要病理特征之一，也是導(dǎo)致視野缺損的主要原因。RGC-5細(xì)胞系是目前作為視神經(jīng)保護(hù)體外實(shí)驗(yàn)?zāi)Ｐ偷囊粋�(gè)重要細(xì)胞系。谷氨酸是視網(wǎng)膜的主要興奮性遞質(zhì)，正常情況下不引起毒性。但在眼壓升高、視神經(jīng)供血不足等導(dǎo)致視網(wǎng)膜缺血缺氧時(shí)，谷氨酸會(huì)大量釋放，對(duì)視網(wǎng)膜神經(jīng)節(jié)細(xì)胞產(chǎn)生毒性作用。谷氨酸的過(guò)度表達(dá)是導(dǎo)致RGCs凋亡的重要原因。因此，利用谷氨酸誘導(dǎo)的RGC-5凋亡可以作為研究視網(wǎng)膜神經(jīng)節(jié)細(xì)胞凋亡及視神經(jīng)保護(hù)的模型。 氯離子是生物體內(nèi)含量最豐富的陰離子，，通過(guò)跨膜轉(zhuǎn)運(yùn)和離子通道參與機(jī)體多種生物功能。電壓門(mén)控性氯離子通道(voltage-gated chloride channnel, ClC)在哺乳動(dòng)物細(xì)胞中廣泛表達(dá)，ClC-2氯離子通道作為此家族中的一個(gè)亞型，是目前研究較為廣泛和明確的一種氯離子通道類型，與細(xì)胞增殖、凋亡及細(xì)胞周期等多種生理功能的調(diào)節(jié)有關(guān)。關(guān)于電壓門(mén)控性氯通道參與細(xì)胞凋亡過(guò)程己得到廣泛證實(shí)。線粒體凋亡通路是細(xì)胞凋亡的主要途徑之一。眼部組織多種細(xì)胞的凋亡都是通過(guò)線粒體凋亡通路完成，并在多種眼科疾病中發(fā)揮作用。Bcl-2基因家族對(duì)細(xì)胞凋亡的激活和調(diào)控起著關(guān)鍵作用，是RGCs主要的內(nèi)源性凋亡通路。 鑒于此，實(shí)驗(yàn)以谷氨酸誘導(dǎo)RGC-5細(xì)胞凋亡作為研究對(duì)象，應(yīng)用cDNA轉(zhuǎn)染和RNAi基因沉默方法分別改變細(xì)胞內(nèi)ClC-2的表達(dá)，觀察細(xì)胞凋亡、細(xì)胞周期變化情況及Bcl-2及Bax蛋白表達(dá)、caspase-3、caspase-9酶活性變化，以探討ClC-2及Bcl-2/Bax調(diào)控的線粒體凋亡通路在此過(guò)程中的作用。 實(shí)驗(yàn)中首先應(yīng)用RT-PCR的方法證實(shí)了RGC-5細(xì)胞中存在ClC-2mRNA的表達(dá)；然后應(yīng)用細(xì)胞免疫熒光染色的方法證實(shí)了ClC-2蛋白在RGC-5細(xì)胞中存在表達(dá)，并定位于細(xì)胞漿，為下一步實(shí)驗(yàn)提供了基礎(chǔ)和依據(jù)。 文獻(xiàn)報(bào)道當(dāng)谷氨酸刺激濃度為lmM時(shí)，并作用于RGC-5細(xì)胞24小時(shí)的凋亡率大約為20%，是較適宜的研究濃度。因此我們將傳代后的細(xì)胞貼壁生長(zhǎng)24小時(shí)后，加入含有l(wèi)mM谷氨酸的無(wú)血清DMEM培養(yǎng)液培養(yǎng)24小時(shí)，從而得到RGC-5細(xì)胞凋亡的模型。 進(jìn)一步實(shí)驗(yàn)以谷氨酸誘導(dǎo)的RGC-5細(xì)胞凋亡為研究對(duì)象，應(yīng)用ClC-2cDNA技術(shù)成功轉(zhuǎn)染細(xì)胞，使細(xì)胞內(nèi)ClC-2的mRNA及蛋白質(zhì)表達(dá)均明顯升高。應(yīng)用MTT法、流式細(xì)胞儀檢測(cè)細(xì)胞的凋亡及周期變化情況。MTT法結(jié)果顯示細(xì)胞的存活率明顯上升，流式細(xì)胞儀檢測(cè)結(jié)果表明凋亡率下降，并且細(xì)胞周期G1期的細(xì)胞比例減少，進(jìn)入S期細(xì)胞增多。以上結(jié)果提示ClC-2的過(guò)表達(dá)對(duì)谷氨酸誘導(dǎo)的RGC-5細(xì)胞凋亡具有保護(hù)作用。 隨后實(shí)驗(yàn)應(yīng)用RNAi技術(shù)轉(zhuǎn)染谷氨酸誘導(dǎo)的RGC-5細(xì)胞，RT-PCR及Westernblot結(jié)果證實(shí)ClC-2的mRNA及蛋白質(zhì)表達(dá)均被明顯抑制，應(yīng)用MTT法、流式細(xì)胞儀檢測(cè)細(xì)胞的凋亡及周期變化情況。MTT法結(jié)果顯示細(xì)胞的存活率明顯下降，流式細(xì)胞儀檢測(cè)結(jié)果表明凋亡率增加，并且細(xì)胞周期G1期的細(xì)胞比例明顯增多，進(jìn)入S期細(xì)胞顯著減少。結(jié)果證實(shí)了抑制ClC-2的表達(dá)可以促進(jìn)細(xì)胞的凋亡。以上兩部分實(shí)驗(yàn)從正反兩方面提示ClC-2氯離子通道對(duì)谷氨酸誘導(dǎo)的RGC-5細(xì)胞凋亡具有保護(hù)作用。 為了進(jìn)一步探討ClC-2氯離子通道如何抑制谷氨酸誘導(dǎo)的RGC-5細(xì)胞凋亡作用，我們進(jìn)行了分子機(jī)制的研究。分別采用cDNA轉(zhuǎn)染技術(shù)增加RGC-5細(xì)胞內(nèi)ClC-2的表達(dá)及RNAi技術(shù)抑制RGC-5細(xì)胞內(nèi)ClC-2的表達(dá)，觀察Bcl-2、Bax蛋白及caspase-3、caspase-9酶活性在各組細(xì)胞中的變化。發(fā)現(xiàn)與谷氨酸誘導(dǎo)組相比，谷氨酸誘導(dǎo)+ClC-2cDNA轉(zhuǎn)染組Bax蛋白表達(dá)、caspase-3、caspase-9酶活性均顯著下降，而B(niǎo)cl-2蛋白表達(dá)顯著升高；相反，與谷氨酸誘導(dǎo)組相比，谷氨酸誘導(dǎo)+ClC-2RNAi組，Bax蛋白表達(dá)、caspase-3、caspase-9酶活性均顯著增高，Bcl-2蛋白表達(dá)顯著降低；以上實(shí)驗(yàn)結(jié)果說(shuō)明了ClC-2氯離子通道的抗凋亡作用是通過(guò)內(nèi)源性線粒體凋亡通路進(jìn)行，并通過(guò)上調(diào)Bcl-2蛋白表達(dá)及下調(diào)Bax蛋白來(lái)調(diào)控細(xì)胞凋亡。 本研究探討了在谷氨酸誘導(dǎo)的RGC-5細(xì)胞凋亡過(guò)程中，ClC-2氯離子通道及線粒體凋亡通路的作用，發(fā)現(xiàn)ClC-2氯離子通道對(duì)谷氨酸誘導(dǎo)的RGC-5細(xì)胞凋亡具有保護(hù)作用，而B(niǎo)cl-2/Bax調(diào)控的線粒體凋亡通路可能參與此過(guò)程。我們的實(shí)驗(yàn)結(jié)果提示ClC-2氯離子通道及Bcl-2/Bax調(diào)控的線粒體凋亡通路可能成為視神經(jīng)保護(hù)的一個(gè)新靶點(diǎn)，為進(jìn)一步研究視神經(jīng)保護(hù)提供了理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。
[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 .
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R77

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