【作者】 于丽；

【导师】 郑雅娟；

【作者基本信息】 吉林大学 ， 眼科学， 2011， 博士

【摘要】 视网膜神经节细胞(retinal ganglion cells, RGCs)的凋亡是青光眼等眼科疾病的重要病理特征之一,也是导致青光眼视野缺损的主要原因。RGC-5细胞系是目前作为青光眼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。但在眼压升高等原因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。电压门控性氯通道(voltage-gated chloride channnel,CIC)在哺乳动物细胞中广泛表达,ClC-3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,与多种生理功能的调节有关。关于ClC参与细胞凋亡过程已得到广泛证实。转化生长因子β(transforming growth factorβ, TGF-β)是一种多功能的细胞间信号蛋白,在眼部多个组织中表达,并在多种眼科疾病中发挥作用。TGF-β言号从细胞膜转入细胞核内而发挥调控基因表达的作用,此过程需要依赖Smads家族蛋白的调节。TGF-β/Smads信号通路在许多临床疾病中发挥作用,也参与了多种细胞的凋亡过程。在哺乳动物中发现Smad蛋白共有8种,其中参与TGF-β信号转导的主要有Smad2、Smad3、Smad4及Smad7。鉴于此,实验以谷氨酸诱导视网膜神经节细胞凋亡作为研究对象,应用cDNA转染和RNAi方法改变细胞内ClC-3的表达,观察细胞凋亡情况及转化生长因子β2、Smads2、Smads3、Smads4、Smads7的表达变化,以探讨ClC-3及TGF-β/Smads信号转导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-2、ClC-3、ClC-5、TGF-β1及TGF-β2 mRNA的表达,并发现TGF-β2表达含量明显高于TGF-β1；然后应用免疫组织化学染色的方法证实了ClC-3蛋白在RGC-5细胞中存在表达,并定位于细胞浆,为进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1mM时,RGC-5细胞的凋亡率大约为20%,是比较适宜的研究浓度,因此我们将传代后的细胞贴壁生长24h后,加入含有1mM谷氨酸的无血清DMEM培养液培养24h,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-3 cDNA技术成功转染细胞,使细胞内ClC-3的mRNA及蛋白质表达均明显升高,应用MTT法、DNA ladder及流式细胞仪检测细胞的凋亡情况。MTT法结果显示细胞的存活率明显上升,DNA ladder及流式细胞仪检测结果表明凋亡率下降,以上结果提示ClC-3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Western blot结果证实ClC-3的mRNA及蛋白质表达均被明显抑制,应用MTT法、DNA ladder及流式细胞仪检测细胞的凋亡情况。MTT法结果显示细胞的存活率明显下降,DNA ladder及流式细胞仪检测结果表明凋亡率明显上升,结果证实了抑制ClC-3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-3氯通道对谷氨酸诱导的RGC-5细胞凋亡具有保护作用。应用RNAi技术抑制ClC-3的表达后,细胞的凋亡率增加,为了进一步检测TGF-β/Smads信号转导通路在上述过程中所起的作用,我们应用RT-PCR及Western blot的方法,检测TGF-β2.Smad2.Smad3.Smad4. Smad7 mRNA及蛋白含量的变化情况,发现TGF-β2及Smad2.Smad3. Smad4.Smad7 mRNA及蛋白表达均有不同程度的增加,提示TGF-p/Smads信号转导通路可能在谷氨酸诱导的RGC-5细胞凋亡过程中发挥作用。本实验探讨了在谷氨酸诱导的RGC-5细胞凋亡过程中,ClC-3氯离子通道及TGF-p/Smad信号通路的作用,并得出结论：ClC-3氯通道对谷氨酸诱导的RGC-5细胞凋亡具有保护作用,而TGF-p/Smad信号通路可能参与此过程。提示ClC-3氯通道及TGF-p/Smad信号通路可能成为视神经保护一个新靶点,为进一步研究青光眼的发病机制及视神经保护提供了理论基础和实验依据。更多还原

【Abstract】 Retinal ganglion cells (RGCs) apoptosis is an important pathological feature of glaucoma and other eye diseases. The RGC-5 cell line is currently used as an in vitro model of glaucoma. Glutamate normally functions as the major excitatory amino acid neurotransmitter in the retina,but at high concentrations it becomes neurotoxic after the intraocular pressure increased,Over-expression of glutamate plays a large role in RGCs apoptosis. Therefore, a glutamate-induced RGC-5 apoptosis model can be used to investigate RGC apoptosis and optic neuroprotection.Chloride ion is the most abundant negion, participating in a variety of biological functions through transmembrane transport and ion channels.Chloride channels (ClC) have been shown extensively distributing in mammal’s organs, tissues and cells.It plays an important role in many physiological and pathological functions,such as apoptosis.The transforming growth factorβ(TGF-β)is a kind of multifunctional protein of cellular signal transmission,which have been shown extensively distributing in eye and play a role in many ophthalmology disease.The TGF-β/Smad signaling pathways play a role in many clinical diseases and are involved in apoptosis in a variety of cells. TGF-βsignaling transfers from the cell membrane to the nucleus and subsequently regulates Smads family proteins. Eight Smad proteins are expressed in mammals, and Smad2, Smad3, Smad4, and Smad7 participate in signal transduction of TGF-β.Reverse transcription (RT)-PCR results revealed ClC-2, ClC-3, and ClC-5,TGF-β1, TGF-β2 mRNA expressed in RGC-5 cells. TGF-p 1 gene expression was significantly less than TGF-β2.Therefore, TGF-β2 represented the detection index in the present study.Immunohistochemical staining suggested that ClC-3 protein expression in RGC-5 cells was localized in the cytoplasm.The apoptosis rate of 20% is a suitable for research at a concentration of 1 mmol/L glutamate. The RGC-5 cells were allowed to grow for 24 hours, followed by culture with serum-free DMEM supplemented with 1 mmol/L glutamate for 24 hours.The present experiment analyzed glutamate-induced RGC-5 cell apoptosis, showing that ClC-3 mRNA and protein expressions significantly increased with ClC-3 cDNA transfection. MTT、DNA ladder assay and flow cytometry analyses detected the cell viability and apoptosis.After the ClC-3 cDNA transfection,MTT assay results demonstrated significantly increased survival rate,flow cytometry and the DNA ladder assay revealed the apoptosis rate was significantly decreased,which suggests that ClC-3 overexpression exhibited a protective effect on apoptosis.Subsequently, ClC-3 mRNA and protein expressions were inhibited by RNAi technology.MTT、DNA ladder assay and flow cytometry analyses detected cell viability and apoptosis.After the ClC-3 expressions were inhibited,MTT assay results demonstrated significantly decreased survival rate,flow cytometry and the DNA ladder assay revealed the apoptosis rate was significantly increased,which suggests that the inhibition of ClC-3 may promote the apoptosis of glutamate-induced RGC-5 cell.The apoptosis rate was significantly increased after the ClC-3 mRNA and protein expressions were inhibited by RNAi technology,then Reverse transcription (RT)-PCR and western blot results revealed Smad2, Smad3, Smad4, and Smad7 mRNA and protein expression increased to varying degrees, suggesting that these molecules contribute to cellular apoptosis.In the present study, overexpression of ClC-3 chloride channel inhibited apoptosis in glutamate-induced RGC-5 cells. Inhibition of ClC-3 chloride channel expression promoted apoptosis, and TGF-β/Smad signaling pathways could play a role in this process. These results demonstrated a role for the ClC-3 chloride channel in TGF-β/Smad signaling pathways, suggesting a novel target for optic nerve and function protection.All my experiments offered new ideas for further study of the pathogenesis of glaucoma and development of neuroprotection.更多还原