發(fā)布時間：2018-03-25 10:22

  本文選題：α晶狀體蛋白　切入點：PAR-2　出處：《第三軍醫(yī)大學(xué)》2012年博士論文

【摘要】：研究背景和意義 視神經(jīng)損傷后視網(wǎng)膜神經(jīng)節(jié)細胞(Retinal ganglion cells, RGCs)的繼發(fā)性凋亡是視功能損傷的病理基礎(chǔ)。課題組前期的系列研究證實從晶狀體分離出來的α晶狀體蛋白對RGCs的存活和突起生長有顯著促進作用，是重要的晶狀體源性“神經(jīng)保護物質(zhì)”。近期研究發(fā)現(xiàn)：損傷狀態(tài)下可誘導(dǎo)內(nèi)源性α晶狀體蛋白表達增加，表達的α晶狀體蛋白主要分布在RGCs膜上，通過抑制Caspase-3活化減緩RGCs凋亡，但其具體作用途徑和機理不明。我們推測，α晶狀體蛋白可能作用于RGCs膜上的某種分子，發(fā)揮生物學(xué)功能。 蛋白酶活化受體-2(protease activated receptors-2，PAR-2)是G蛋白耦聯(lián)受體(Gproteincoupled receptors, GPCRs)家族中的一員，被激活后可參與多種病理生理過程。研究發(fā)現(xiàn)，大鼠眼在發(fā)育過程中或視神經(jīng)損傷后，視網(wǎng)膜中PAR-2mRNA表達明顯上調(diào)，提示PAR-2可能與眼或視神經(jīng)的發(fā)育再生有關(guān)。有文獻報道，PAR-2可與α晶狀體蛋白結(jié)合并表達于星形膠質(zhì)細胞膜上，并且PAR-2活化對α晶狀體蛋白抗細胞凋亡效應(yīng)具有協(xié)同作用。 PAR-2作為一種重要的G蛋白耦聯(lián)膜受體，那么它在RGCs中的表達及功能如何？它與α晶狀體蛋白在RGCs中是否存在相互作用關(guān)系？PAR-2是否是α晶狀體蛋白發(fā)揮抗RGCs凋亡的關(guān)鍵的起始分子？以上問題目前尚不清楚。因此，進一步探討內(nèi)源性α晶狀體蛋白保護RGCs的機制，將為α晶狀體蛋白基因治療或合成新型的短肽藥物奠定理論基礎(chǔ)。 目的 本研究擬利用在體和離體損傷模型觀察膜受體PAR-2在RGCs中的表達與功能；明確α晶狀體蛋白與PAR-2的相互作用關(guān)系，從分子水平上求證α晶狀體蛋白是否通過促進PAR-2活化，發(fā)揮了胞內(nèi)抗細胞凋亡的作用，本研究是課題組前期工作的繼續(xù)和深入，為探索治療視神經(jīng)損傷的新策略奠定理論基礎(chǔ)。 方法 第二章 1．建立Long Evans大鼠視神經(jīng)鉗夾傷在體模型，于傷后第1、3、7、14、28天行視網(wǎng)膜冰凍切片，采用免疫熒光染色觀察傷后不同時點視網(wǎng)膜中PAR-2的表達分布情況；建立大鼠RGC-5缺氧損傷離體模型，采用熒光定量PCR和western blot檢測缺氧培養(yǎng)6、12、24、48h不同時點RGC-5細胞內(nèi)PAR-2的表達規(guī)律。 2．建立大鼠RGC-5細胞缺氧損傷離體模型，激活或抑制PAR-2，采用CCK-8檢測細胞的存活率；Hochest33342染色定性觀察細胞凋亡情況；Annexin V-FITC流式細胞術(shù)定量檢測細胞凋亡率，多角度觀察PAR-2活化對缺氧誘導(dǎo)RGCs凋亡的影響；其次進一步通過Western Blot法檢測Bcl-2、Bax及Caspase-3的蛋白表達水平，初步探討可能涉及的凋亡途徑。 第三章 1．建立視神經(jīng)鉗夾傷在體模型，于傷后第1、3、7、14、28天：①行視網(wǎng)膜冰凍切片，采用免疫熒光雙染觀察傷后不同時點α晶狀體蛋白與PAR-2在視網(wǎng)膜中的表達與定位；②提取視網(wǎng)膜組織，免疫共沉淀法進一步檢測α晶狀體蛋白與PAR-2在視網(wǎng)膜中的相互作用。 2．建立RGC-5細胞缺氧損傷離體模型，于缺氧培養(yǎng)6、12、24、48h后：①采用免疫熒光雙染檢測缺氧不同時點α晶狀體蛋白與PAR-2在RGC-5細胞中的表達與定位；②收集細胞，免疫共沉淀法檢測兩者在RGC-5細胞中的相互作用。 第四章 1．基于已知的α晶狀體蛋白與PAR-2相互作用的氨基酸序列，以本實驗室備存的野生型α晶狀體蛋白重組腺病毒表達載體為模板，采用分子克隆常規(guī)技術(shù)，構(gòu)建120～154氨基酸缺失的突變型α晶狀體蛋白重組腺病毒表達載體并鑒定。 2．將構(gòu)建好的突變型α晶狀體蛋白重組腺病毒與已有的野生型α晶狀體蛋白重組腺病毒分別轉(zhuǎn)染至目的細胞RGC-5，Western Blot明確兩者在RGC-5細胞中的表達情況，免疫共沉淀法鑒定兩者與PAR-2的結(jié)合能力。 3．在此基礎(chǔ)上采用Hochest33342染色和Annexin V-FITC流式細胞術(shù)檢測細胞凋亡情況，鈣探針Rhod-2/AM檢測細胞內(nèi)PAR-2依賴的鈣瞬變情況，對比分析野生型和突變型α晶狀體蛋白轉(zhuǎn)染對缺氧誘導(dǎo)的RGC-5細胞的凋亡及PAR-2活性的影響，明確α晶狀體蛋白是否通過促進PAR-2活化，發(fā)揮了胞內(nèi)抗細胞凋亡的作用。 結(jié)果 第二章 1．在體實驗發(fā)現(xiàn)，成年大鼠正常視網(wǎng)膜的內(nèi)核層、內(nèi)叢狀層及節(jié)細胞層可見少量PAR-2的表達，視神經(jīng)損傷后，PAR-2的分布范圍擴大且表達增強，傷后7d和14d達高峰，28d時明顯下降；離體實驗也證實正常培養(yǎng)的RGC-5細胞有PAR-2mRNA和蛋白水平表達，缺氧損傷后表達明顯增強，mRNA水平在12h達高峰，而蛋白水平在24h達高峰，缺氧48h均下降至正常水平。 2．CCK-8、Hochest33342染色和Annexin V-FITC流式細胞術(shù)等結(jié)果一致證實PAR-2活化能增加缺氧損傷下RGC-5細胞的存活率，減緩細胞凋亡的程度。 3．Western Blot法進一步明確PAR-2活化后可通過增加抗凋亡蛋白Bcl-2的表達，減少促凋亡蛋白Bax的表達，降低Caspase-3的活化，達到促進細胞存活的目的。 第三章 1．在體實驗發(fā)現(xiàn)，正常成年大鼠視網(wǎng)膜的內(nèi)核層、內(nèi)叢狀層及視網(wǎng)膜節(jié)細胞層可見少量PAR-2的陽性染色，而視網(wǎng)膜各層均未見明顯的α晶狀體蛋白陽性染色，兩者在節(jié)細胞層未見共表達，免疫共沉淀實驗表明無相互作用；視神經(jīng)損傷后，PAR-2和α晶狀體蛋白在視網(wǎng)膜中的分布范圍擴大且表達增強，于傷后7d達高峰，在節(jié)細胞層有明顯的共表達，免疫共沉淀實驗進一步表明兩者在視神經(jīng)損傷后視網(wǎng)膜組織中存在相互作用。 2．離體實驗發(fā)現(xiàn)，正常培養(yǎng)的RGC-5細胞膜上可見少量PAR-2的陽性染色，而幾乎未見α晶狀體蛋白的陽性染色，兩者在常氧狀態(tài)下的RGC-5細胞上未見共表達，免疫共沉淀表明兩者無相互作用。RGC-5細胞缺氧損傷后，PAR-2和α晶狀體蛋白的表達增強，12h和24h達高峰，48h明顯下降。兩者在RGC-5細胞膜上可見共表達，免疫共沉淀實驗進一步表明損傷狀態(tài)下兩者存在相互作用。 第四章 1．蛋白電泳及基因測序一致證實成功構(gòu)建了120～154氨基酸缺失的突變型α晶狀體蛋白重組腺病毒。 2．將野生型和突變型α晶狀體蛋白重組腺病毒分別轉(zhuǎn)染至RGC-5細胞中，WesternBlot結(jié)果顯示常氧及缺氧損傷后均可實現(xiàn)目的基因的過表達。免疫共沉淀進一步對比證實野生型α晶狀體蛋白能結(jié)合PAR-2，而突變型α晶狀體蛋白失去與PAR-2結(jié)合的能力。 3．將野生型及突變型α晶狀體蛋白重組腺病毒分別轉(zhuǎn)染至RGC-5細胞中，，Hochest33342染色和Annexin V-FITC流式細胞術(shù)一致證實過表達的野生型α晶狀體蛋白能明顯減緩缺氧誘導(dǎo)RGC-5細胞的凋亡，而突變型α晶狀體蛋白保護作用與野生型α晶狀體蛋白相比明顯減弱，兩組實驗結(jié)果有統(tǒng)計學(xué)差異。與此同時鈣瞬變實驗進一步顯示，野生型α晶狀體蛋白能增加缺氧損傷后RGC-5細胞內(nèi)PAR-2依賴的鈣瞬變幅值，表明能促進PAR-2的活化，而突變型α晶狀體蛋白對鈣瞬變幅值無影響。以上結(jié)果表明α晶狀體蛋白發(fā)揮抗細胞凋亡的作用與結(jié)合PAR-2后促其活化有關(guān)。 全文結(jié)論 1．離體及在體實驗證實，正常大鼠視網(wǎng)膜神經(jīng)節(jié)細胞上有膜受體PAR-2的表達，損傷可誘導(dǎo)其表達上調(diào)。 2．損傷條件下PAR-2活化能增加視網(wǎng)膜神經(jīng)節(jié)細胞的存活，減緩凋亡的程度，其保護作用與增加Bcl-2/Bax的比值，降低Caspase-3的活化有關(guān)，提示線粒體凋亡途徑可能參與其中。 3．離體及在體實驗證實，損傷可誘導(dǎo)視網(wǎng)膜神經(jīng)節(jié)細胞上內(nèi)源性α晶狀體蛋白與膜受體PAR-2的表達上調(diào)，且兩者存在相互作用關(guān)系。 4．損傷條件下α晶狀體蛋白可通過結(jié)合膜受體PAR-2并促其活化，發(fā)揮抑制視網(wǎng)膜神經(jīng)節(jié)細胞凋亡的作用，初步揭示α晶狀體蛋白發(fā)揮神經(jīng)保護作用的分子機制。
[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.
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