氯离子通道ClC-2对小梁细胞骨架结构作用的实验研究

发布时间：2018-07-07 18:02

本文选题：氯离子通道ClC-2 + 青光眼　；参考：《吉林大学》2014年博士论文

【摘要】：原发性开角型青光眼（Primary Open-Angle Glaucoma，POAG）是由于眼内压升高而引起的一种不可逆的致盲眼病，其致病机制与小梁细胞形态、骨架结构及生理功能密切相关。近期相关文献报道小梁细胞骨架结构发生异常改变，能引起眼压升高，有可能诱发原发性开角型青光眼。氯离子通道家族具有调控生物体内细胞的兴奋度、跨上皮细胞内的物质转运、pH值及生物体内细胞容积等多种功能，另外还对细胞分化、细胞增生、细胞凋亡及免疫应答等生理功能有着一定的调控作用。电压门控性氯离子通道(Voltage-Gated Chloride Channel，简称ClC)是氯离子通道家族的一员，目前共发现9种ClC型氯离子通道，ClC-2是ClC型氯离子通道中的一种。本项目组已证实ClC-2存在于小梁细胞，并且能调控小梁细胞多项生理活动。本实验应用RNA干扰技术，观察ClC-2表达与小梁细胞骨架结构的关系，并探讨ClC-2对小梁细胞骨架结构影响的作用机制。方法： 1）ClC-2-siRNA设计、合成及活性筛选：根据ClC-2基因mRNA序列，利用计算机相关软件设计合成ClC-2-siRNA，利用Realtime-PCR，Western Blot技术检测ClC-2-siRNA对ClC-2表达的影响。 2）ClC-2对人小梁细胞骨架的影响：利用Real time-PCR，WesternBlot及免疫荧光技术检测ClC-2-siRNA对Actin，Vinculin，β-catenin表达的影响。 3）ClC-2对人小梁细胞骨架结构影响的机制研究：构建人小梁细胞体外青光眼模型；利用Real time-PCR，Western Blot检测体外青光眼模型小梁细胞ClC-2的表达；利用Western Blot检测TGF-β/Smad信号转导通路的表达。结果： 1）本实验引进美国ScienCell公司的正常人小梁细胞作为实验细胞株，，并且应用了相应的成纤维细胞培养基，细胞单层螺旋状排列，为梭形，生长良好，成功进行了传代及冻存，为后续实验奠定了良好基础。由于目前没有特异的针对ClC-2的阻断剂,为了研究ClC-2的活性与小梁细胞骨架的关系，因此我们应用了RNAi干扰技术。根据ClC-2基因序列和RNAi的设计原则，应用siRNA设计软件，分别设计了ClC-2-siRNA1，ClC-2-siRNA2.同时本文针对ClC-2-siRNA1设计了反相siRNA(scRNA)作为对照。结果证实ClC-2-siRNA最佳的转染浓度是100pmol，最佳作用时间是转染后24h。ClC-2-siRNA1对ClC-2mRNA及蛋白表达的抑制效果优于ClC-2-siRNA2。 2）小梁细胞骨架的正常结构是维持小梁细胞功能的重要基础。我们主要研究ClC-2活性与小梁细胞骨架结构是否存在某种关联，是否与原发性开角型青光眼的发病机制有关系？肌动蛋白和粘着斑蛋白是小梁细胞主要的骨架蛋白，β-链蛋白是一种黏附因子，能增加细胞之间的粘合程度。我们用免疫荧光染色技术、RT-PCR、Western Blot的方法对比了正常小梁细胞和转染后的小梁细胞骨架的变化，以及肌动蛋白、粘着斑蛋白和β-链蛋白表达变化。结果发现转染后的小梁细胞的细胞骨架受到了一定的影响，细胞骨架的肌动蛋白发生紊乱，肌动蛋白肌丝出现了扭曲交联，微丝虽然增加但失去了正常的排列。黏着斑蛋白排列发生了变化，无法沿着肌动蛋白微丝进行有序排列。同时转染后的小梁细胞的肌动蛋白、粘着斑蛋白和β-链蛋白的蛋白含量、mRNA表达较正常增加，差异有统计学意义。 3）小梁细胞体外青光眼模型的细胞形态与正常小梁细胞区别不大，主要是细胞体积略大，核偏圆，排列略显不规则。我们应用RT-PCR、Western Blot的方法检测体外青光眼模型小梁细胞的ClC-2的表达较正常小梁细胞下降，差异具有统计学意义；其肌动蛋白、粘着斑蛋白和β-链蛋白的蛋白含量、mRNA表达较正常增加，差异具有统计学意义。转化生长因子（TGF-β）在POAG发病机制中起着重要的作用，为了探讨ClC-2活性对小梁细胞骨架影响的机制，我们应用RT-PCR、Western Blot方法检测了ClC-2-siRNA转染后的小梁细胞TGF-β和Smad2的表达。结果发现ClC-2-siRNA转染后的小梁细胞的TGF-β和Smad2的表达较正常小梁细胞增加，差异有统计学意义，推测TGF-β/Smad转导通路可能在ClC-2对小梁细胞骨架影响过程中发挥了重要作用。结论： 1）ClC-2对人小梁细胞骨架结构具有保护作用。 2）ClC-2对人小梁细胞骨架结构具有的保护作用可能与TGF-β/Smad信号转导通路有关
[Abstract]:Primary Open - Angle glaucoma ( POAG ) is an irreversible blindness caused by elevated intraocular pressure . The pathogenesis of primary open - angle glaucoma is closely related to the morphology , skeletal structure and physiological function of trabecular cells . Recently , there have been reported abnormal changes in the cytoskeletal structure of small beam , which can cause elevation of intraocular pressure , and may induce primary open - angle glaucoma .

ClC - 2 is a member of chloride channel family . It has been found that ClC - 2 is a member of chloride channel family .

Method :

1 ) ClC - 2 - siRNA design , synthesis and activity screening : According to ClC - 2 gene mRNA sequence , ClC - 2 - siRNA was synthesized by computer - related software design , and the effects of ClC - 2 - siRNA on ClC - 2 expression were detected by using Realtime - PCR and Western Blot .

2 ) The effect of ClC - 2 on the cytoskeletal structure of human trabecular : The effects of ClC - 2 - siRNA on the expression of Actin , 尾 - catenin were detected by Real time - PCR , Western Blot and immunofluorescence technique .

3 ) The mechanism of ClC - 2 on the structure of human trabecular cells : A model of human trabecular cell in vitro glaucoma was constructed .
The expression of ClC - 2 was detected by Real time - PCR and Western Blot .
The expression of TGF - 尾 / Smad signal transduction pathway was detected by Western Blot .

Results :

In order to study the relationship between ClC - 2 - siRNA1 and ClC - 2 - siRNA1 , ClC - 2 - siRNA1 and ClC - 2 - siRNA2 were designed .

2 ) The normal structure of small - beam cytoskeletal structure is an important basis for maintaining the function of small - beam cells . We mainly study the relationship between ClC - 2 activity and the pathogenesis of primary open - angle glaucoma .

3 ) The cell morphology of trabecular cell in vitro was not significantly different from that of normal trabecular cells . It was found that the expression of ClC - 2 in trabecular cells in vitro was significantly smaller than that in normal trabecular cells by RT - PCR and Western Blot .
The expression of TGF - 尾 and Smad2 in small - beam cells transfected with ClC - 2 - siRNA was detected by RT - PCR and Western Blot .

Conclusion :

1 ) ClC - 2 has protective effect on the cytoskeletal structure of human trabecular cell .

2 ) The protective effect of ClC - 2 on the cytoskeletal structure of human trabecular cells may be related to TGF - 尾 / Smad signal transduction pathways .
【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】：R775.2

【参考文献】