PAR-2在α晶状体蛋白保护视网膜神经节细胞中的作用研究

发布时间：2018-03-25 10:22

  本文选题：α晶状体蛋白　切入点：PAR-2　出处：《第三军医大学》2012年博士论文

【摘要】：研究背景和意义 视神经损伤后视网膜神经节细胞(Retinal ganglion cells, RGCs)的继发性凋亡是视功能损伤的病理基础。课题组前期的系列研究证实从晶状体分离出来的α晶状体蛋白对RGCs的存活和突起生长有显著促进作用，是重要的晶状体源性“神经保护物质”。近期研究发现：损伤状态下可诱导内源性α晶状体蛋白表达增加，表达的α晶状体蛋白主要分布在RGCs膜上，通过抑制Caspase-3活化减缓RGCs凋亡，但其具体作用途径和机理不明。我们推测，α晶状体蛋白可能作用于RGCs膜上的某种分子，发挥生物学功能。 蛋白酶活化受体-2(protease activated receptors-2，PAR-2)是G蛋白耦联受体(Gproteincoupled receptors, GPCRs)家族中的一员，被激活后可参与多种病理生理过程。研究发现，大鼠眼在发育过程中或视神经损伤后，视网膜中PAR-2mRNA表达明显上调，提示PAR-2可能与眼或视神经的发育再生有关。有文献报道，PAR-2可与α晶状体蛋白结合并表达于星形胶质细胞膜上，并且PAR-2活化对α晶状体蛋白抗细胞凋亡效应具有协同作用。 PAR-2作为一种重要的G蛋白耦联膜受体，那么它在RGCs中的表达及功能如何？它与α晶状体蛋白在RGCs中是否存在相互作用关系？PAR-2是否是α晶状体蛋白发挥抗RGCs凋亡的关键的起始分子？以上问题目前尚不清楚。因此，进一步探讨内源性α晶状体蛋白保护RGCs的机制，将为α晶状体蛋白基因治疗或合成新型的短肽药物奠定理论基础。 目的 本研究拟利用在体和离体损伤模型观察膜受体PAR-2在RGCs中的表达与功能；明确α晶状体蛋白与PAR-2的相互作用关系，从分子水平上求证α晶状体蛋白是否通过促进PAR-2活化，发挥了胞内抗细胞凋亡的作用，本研究是课题组前期工作的继续和深入，为探索治疗视神经损伤的新策略奠定理论基础。 方法 第二章 1．建立Long Evans大鼠视神经钳夹伤在体模型，于伤后第1、3、7、14、28天行视网膜冰冻切片，采用免疫荧光染色观察伤后不同时点视网膜中PAR-2的表达分布情况；建立大鼠RGC-5缺氧损伤离体模型，采用荧光定量PCR和western blot检测缺氧培养6、12、24、48h不同时点RGC-5细胞内PAR-2的表达规律。 2．建立大鼠RGC-5细胞缺氧损伤离体模型，激活或抑制PAR-2，采用CCK-8检测细胞的存活率；Hochest33342染色定性观察细胞凋亡情况；Annexin V-FITC流式细胞术定量检测细胞凋亡率，多角度观察PAR-2活化对缺氧诱导RGCs凋亡的影响；其次进一步通过Western Blot法检测Bcl-2、Bax及Caspase-3的蛋白表达水平，初步探讨可能涉及的凋亡途径。 第三章 1．建立视神经钳夹伤在体模型，于伤后第1、3、7、14、28天：①行视网膜冰冻切片，采用免疫荧光双染观察伤后不同时点α晶状体蛋白与PAR-2在视网膜中的表达与定位；②提取视网膜组织，免疫共沉淀法进一步检测α晶状体蛋白与PAR-2在视网膜中的相互作用。 2．建立RGC-5细胞缺氧损伤离体模型，于缺氧培养6、12、24、48h后：①采用免疫荧光双染检测缺氧不同时点α晶状体蛋白与PAR-2在RGC-5细胞中的表达与定位；②收集细胞，免疫共沉淀法检测两者在RGC-5细胞中的相互作用。 第四章 1．基于已知的α晶状体蛋白与PAR-2相互作用的氨基酸序列，以本实验室备存的野生型α晶状体蛋白重组腺病毒表达载体为模板，采用分子克隆常规技术，构建120～154氨基酸缺失的突变型α晶状体蛋白重组腺病毒表达载体并鉴定。 2．将构建好的突变型α晶状体蛋白重组腺病毒与已有的野生型α晶状体蛋白重组腺病毒分别转染至目的细胞RGC-5，Western Blot明确两者在RGC-5细胞中的表达情况，免疫共沉淀法鉴定两者与PAR-2的结合能力。 3．在此基础上采用Hochest33342染色和Annexin V-FITC流式细胞术检测细胞凋亡情况，钙探针Rhod-2/AM检测细胞内PAR-2依赖的钙瞬变情况，对比分析野生型和突变型α晶状体蛋白转染对缺氧诱导的RGC-5细胞的凋亡及PAR-2活性的影响，明确α晶状体蛋白是否通过促进PAR-2活化，发挥了胞内抗细胞凋亡的作用。 结果 第二章 1．在体实验发现，成年大鼠正常视网膜的内核层、内丛状层及节细胞层可见少量PAR-2的表达，视神经损伤后，PAR-2的分布范围扩大且表达增强，伤后7d和14d达高峰，28d时明显下降；离体实验也证实正常培养的RGC-5细胞有PAR-2mRNA和蛋白水平表达，缺氧损伤后表达明显增强，mRNA水平在12h达高峰，而蛋白水平在24h达高峰，缺氧48h均下降至正常水平。 2．CCK-8、Hochest33342染色和Annexin V-FITC流式细胞术等结果一致证实PAR-2活化能增加缺氧损伤下RGC-5细胞的存活率，减缓细胞凋亡的程度。 3．Western Blot法进一步明确PAR-2活化后可通过增加抗凋亡蛋白Bcl-2的表达，减少促凋亡蛋白Bax的表达，降低Caspase-3的活化，达到促进细胞存活的目的。 第三章 1．在体实验发现，正常成年大鼠视网膜的内核层、内丛状层及视网膜节细胞层可见少量PAR-2的阳性染色，而视网膜各层均未见明显的α晶状体蛋白阳性染色，两者在节细胞层未见共表达，免疫共沉淀实验表明无相互作用；视神经损伤后，PAR-2和α晶状体蛋白在视网膜中的分布范围扩大且表达增强，于伤后7d达高峰，在节细胞层有明显的共表达，免疫共沉淀实验进一步表明两者在视神经损伤后视网膜组织中存在相互作用。 2．离体实验发现，正常培养的RGC-5细胞膜上可见少量PAR-2的阳性染色，而几乎未见α晶状体蛋白的阳性染色，两者在常氧状态下的RGC-5细胞上未见共表达，免疫共沉淀表明两者无相互作用。RGC-5细胞缺氧损伤后，PAR-2和α晶状体蛋白的表达增强，12h和24h达高峰，48h明显下降。两者在RGC-5细胞膜上可见共表达，免疫共沉淀实验进一步表明损伤状态下两者存在相互作用。 第四章 1．蛋白电泳及基因测序一致证实成功构建了120～154氨基酸缺失的突变型α晶状体蛋白重组腺病毒。 2．将野生型和突变型α晶状体蛋白重组腺病毒分别转染至RGC-5细胞中，WesternBlot结果显示常氧及缺氧损伤后均可实现目的基因的过表达。免疫共沉淀进一步对比证实野生型α晶状体蛋白能结合PAR-2，而突变型α晶状体蛋白失去与PAR-2结合的能力。 3．将野生型及突变型α晶状体蛋白重组腺病毒分别转染至RGC-5细胞中，，Hochest33342染色和Annexin V-FITC流式细胞术一致证实过表达的野生型α晶状体蛋白能明显减缓缺氧诱导RGC-5细胞的凋亡，而突变型α晶状体蛋白保护作用与野生型α晶状体蛋白相比明显减弱，两组实验结果有统计学差异。与此同时钙瞬变实验进一步显示，野生型α晶状体蛋白能增加缺氧损伤后RGC-5细胞内PAR-2依赖的钙瞬变幅值，表明能促进PAR-2的活化，而突变型α晶状体蛋白对钙瞬变幅值无影响。以上结果表明α晶状体蛋白发挥抗细胞凋亡的作用与结合PAR-2后促其活化有关。 全文结论 1．离体及在体实验证实，正常大鼠视网膜神经节细胞上有膜受体PAR-2的表达，损伤可诱导其表达上调。 2．损伤条件下PAR-2活化能增加视网膜神经节细胞的存活，减缓凋亡的程度，其保护作用与增加Bcl-2/Bax的比值，降低Caspase-3的活化有关，提示线粒体凋亡途径可能参与其中。 3．离体及在体实验证实，损伤可诱导视网膜神经节细胞上内源性α晶状体蛋白与膜受体PAR-2的表达上调，且两者存在相互作用关系。 4．损伤条件下α晶状体蛋白可通过结合膜受体PAR-2并促其活化，发挥抑制视网膜神经节细胞凋亡的作用，初步揭示α晶状体蛋白发挥神经保护作用的分子机制。
[Abstract]:Research background and significance
After optic nerve injury of retinal ganglion cells (Retinal ganglion cells, RGCs) of the secondary apoptosis is the pathological basis of visual function damage. A series of research group of early confirmed isolated from lens alpha crystallin on survival and neurite growth of RGCs has a significant role in promoting, is an important source of neuroprotective substances "lens". Recent research found that the damage state can induce endogenous alpha crystallin expression increased the expression of alpha crystallin is mainly distributed in the RGCs membrane, inhibit Caspase-3 activity, slow RGCs apoptosis, but the concrete mechanism and the mechanism is unknown. We hypothesized that a molecule alpha crystallin protein may play a role in the RGCs film on the play biological function.
Protease activated receptor -2 (protease activated receptors-2, PAR-2) is a G protein coupled receptors (Gproteincoupled, receptors, GPCRs) is a member of the family, is activated may participate in a variety of physiological and pathological processes. The study found that in rat eyes during development or optic nerve injury, up-regulated the expression of PAR-2mRNA in retina, suggesting that the development of regeneration of PAR-2 may be related to eye or optic nerve. There are reports in the literature, PAR-2 can be combined with alpha crystallin and expressed in astrocytic cell membrane, and PAR-2 on activation of alpha crystallin anti apoptosis effect has a synergistic effect.
PAR-2 as an important G protein coupled receptors, so it expression and function in RGCs? With alpha crystallin in RGCs interaction relationship exists? Whether PAR-2 is alpha crystallin play anti apoptosis of RGCs key molecules to ask questions before starting? Above is not clear. Therefore, to further investigate the effect of endogenous alpha crystallin RGCs protection mechanism, will provide a theoretical basis for the alpha crystallin gene therapy or synthesis of novel peptide drugs.
objective
This study intends to use the in vivo and in vitro to observe the injury model of membrane receptor PAR-2 in RGCs expression and function; clear interaction between alpha crystallin and PAR-2, from the molecular level to verify whether the alpha crystallin can promote PAR-2 activation, played anti apoptosis effect, this study is a continuation of the preliminary work the research group and in-depth, lay the theoretical foundation for the exploration of new strategies for the treatment of optic nerve injury.
Method
The second chapter
1. the establishment of Long Evans of rat optic nerve injury in vivo. 1,3,7,14,28 days after injury in the retinal sections. Immunofluorescence staining was used to observe the injury at different time points after PAR-2 expression in retina distribution; establish hypoxia rat RGC-5 damage in vitro model, using fluorescence quantitative PCR and Western blot detection of hypoxia 6,12,24,48h at the same time point RGC-5 intracellular PAR-2 expression patterns.
2. the establishment of hypoxic rat RGC-5 cell injury model in vitro, activate or inhibit PAR-2, the survival rate of cells was detected by CCK-8 assay; Hochest33342 staining to observe the apoptosis of Annexin V-FITC cells; flow cytometry quantitative detection of apoptosis rate, multi angle observation of PAR-2 activation effect on hypoxia induced apoptosis of RGCs; then further detected by Bcl-2 Western Blot method, the expression level of Bax and Caspase-3 protein, preliminary study of apoptosis pathway may be involved.
The third chapter
1. the establishment of optic nerve injury in the model, in the 1,3,7,14,28 days after injury of retinal frozen sections, expression and localization by immunofluorescence double staining were observed at different time points after alpha crystallin and PAR-2 in the retina; the extraction of retinal tissue, CO immunoprecipitation method was further used to detect interaction of alpha crystallin and PAR-2 in the retina.
2. RGC-5 cell hypoxia injury in vitro model to hypoxia after 6,12,24,48h: double immunofluorescence staining for the expression and localization of hypoxia and alpha crystallin and PAR-2 in RGC-5 cells by 1; the collection of cells, CO immunoprecipitation interaction was detected both in RGC-5 cells.
The fourth chapter
1. based on the amino acid sequence of alpha crystallin and known PAR-2 interaction, expression vector as a template in the laboratory is kept by the wild-type alpha crystallin recombinant adenovirus, cloning technology using conventional molecular construction, 120~154 amino acid deletion mutant alpha crystallin protein recombinant adenovirus expression vector and identification.
2. the structure of wild type alpha crystallin recombinant adenovirus mutant recombinant adenovirus and alpha crystallin has built was transfected to RGC-5 cells, the expression of Western Blot clearly both in RGC-5 cells, with CO immunoprecipitation method to identify them and PAR-2 ability.
3锛庡湪姝ゅ熀纭

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