本文關(guān)鍵詞：過表達(dá)Abba1重組表達(dá)質(zhì)粒對COS7細(xì)胞膜結(jié)構(gòu)影響的實(shí)驗(yàn)研究　出處：《南京醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 細(xì)胞膜結(jié)構(gòu) Abba1 IMD ruffling Rac1

【摘要】：背景和目的:細(xì)胞運(yùn)動(dòng)是人類生命活動(dòng)中重要的病理生理基礎(chǔ),與衰老、血管新生和腫瘤轉(zhuǎn)移密切相關(guān)。細(xì)胞膜結(jié)構(gòu)重塑是細(xì)胞運(yùn)動(dòng)的重要結(jié)構(gòu)基礎(chǔ),主要包括胞膜突起、凹陷和管狀結(jié)構(gòu)的形成。在胞膜突起結(jié)構(gòu)中根據(jù)形態(tài)和直徑大小可以大致分為microspikes(微刺狀)和ruffling(皺褶狀)結(jié)構(gòu)。目前的研究認(rèn)為,IMD (IRSp53/MIM homology domain)蛋白質(zhì)家族參與細(xì)胞膜結(jié)構(gòu)重塑。該蛋白質(zhì)家族共有一段高度保守的IMD結(jié)構(gòu)域,其中一個(gè)家族成員是Abba1。體外實(shí)驗(yàn)中將Abba1和Abba1-IMD分別轉(zhuǎn)染細(xì)胞,發(fā)現(xiàn)過表達(dá)全長的Abba1主要促進(jìn)細(xì)胞ruffling生成,而轉(zhuǎn)染單獨(dú)的IMD主要促進(jìn)細(xì)胞microspikes的生成。本實(shí)驗(yàn)旨在初步探討Abba1氨基酸序列中調(diào)節(jié)細(xì)胞膜結(jié)構(gòu)重塑的結(jié)構(gòu)域。 方法:本實(shí)驗(yàn)將全長的hAbba1 (human Abba1,人源Abba1)序列或hAbba1的序列片段分別載入表達(dá)GFP綠色熒光蛋白的表達(dá)載體pEGFP中,形成Abba1的基因重組表達(dá)質(zhì)粒,形成Abba1的重組表達(dá)質(zhì)粒。 包括: 4177(pEGFP-hAbba1-del-WH2) 4178(pEGFP-hAbba1-IMD) 4210(pEGFP-hAbba1-del-SRD) 4764(pEGFP-hIRSp53-IMD-hAbba1-SRD1-SRD2-WH2) 4842(pEGFP-hAbba1-IMD-hIRSp53-SH3-WH2-like)5160(pEGFP-hAbba1-IMD-(Lys13Glu13)-SRD1-SRD2-WH2) 5244(pEGFP-hAbba1-IMD-SRD1-SRD2-(SerAla)-WH2) 5231(pEGFP-hAbba1-del-357-553aa-WH2) 5232(pEGFP-hAbba1-del-357-722aa-WH2) 5399(pEGFP-hAbba1-del-554-722aa-WH2)。 雙酶切和基因測序驗(yàn)證上述Abba1基因重組表達(dá)質(zhì)粒。將制備成功的重組表達(dá)質(zhì)粒分別瞬時(shí)轉(zhuǎn)染COS7細(xì)胞,通過免疫熒光將GFP綠色熒光蛋白染色從而示蹤顯示轉(zhuǎn)染成功的COS7細(xì)胞,并在顯微鏡下觀察細(xì)胞膜結(jié)構(gòu)的改變。 結(jié)果:免疫熒光結(jié)果顯示:同GFP對照組相比,過表達(dá)全長的Abba1主要促進(jìn)細(xì)胞ruffling生成,與轉(zhuǎn)染全長Abba1產(chǎn)生類似的細(xì)胞膜突起結(jié)構(gòu)的基因重組表達(dá)質(zhì)粒還有4764(pEGFP-hIRSp53-IMD-hAbba1-SRD1-SRD2-WH2), 4177(pEGFP-hAbba1-del-WH2),4210(pEGFP-hAbba1-del-SRD),5244(pEGFP-hAbba1-IMD-SRD1-SRD2-(SerAla)-WH2),5231(pEGFP-hAbba1-del-357-553aa-WH2), 5399(pEGFP-hAbba1-del-554-722aa-WH2);與轉(zhuǎn)染全長Abba1的結(jié)果相反,過表達(dá)5160(pEGFP-hAbba1-IMD-(Lys13Glu13)-SRD1-SRD2-WH2)的COS7細(xì)胞基本不形成ruffling;比較特殊的是轉(zhuǎn)染4178(pEGFP-hAbba1-IMD)的COS7細(xì)胞基本不形成ruffling,而是主要促進(jìn)細(xì)胞microspikes的生成。類似的結(jié)果還發(fā)生在過表達(dá)基因重組表達(dá)質(zhì)粒4842(pEGFP-hAbba1-IMD-hIRSp53-SH3-WH2-like)和5232(pEGFP-hAbba1-del-357-722aa-WH2)的COS7細(xì)胞中。 研究證實(shí)Abba1的IMD能與Rac1可以相互作用,并通過上調(diào)GTP-Rac1(Rac1的活性形式)的蛋白水平促進(jìn)細(xì)胞產(chǎn)生ruffling。本實(shí)驗(yàn)發(fā)現(xiàn),與對照組相比,過表達(dá)全長Abba1的COS7細(xì)胞中GTP-Rac1蛋白水平升高(P 0.05),而過表達(dá)4178(pEGFP-hAbba1-IMD)和5232(pEGFP-hAbba1-del-357-722aa-WH2)的COS7細(xì)胞中GTP-Rac1蛋白水平降低(P 0.05)。 結(jié)論:Abba1單獨(dú)的IMD主要促進(jìn)細(xì)胞microspikes的生成。在全長的Abba1序列中,IMD又參與調(diào)節(jié)細(xì)胞產(chǎn)生ruffling,其13個(gè)賴氨酸為主要的功能位點(diǎn)。因此IMD是調(diào)節(jié)不同細(xì)胞膜結(jié)構(gòu)重塑的重要功能域。Abba1的357-722aa這段氨基酸序列對細(xì)胞ruffling的產(chǎn)生也很關(guān)鍵,而且實(shí)驗(yàn)結(jié)果提示該區(qū)域參與Abba1介導(dǎo)細(xì)胞產(chǎn)生ruffling的功能位點(diǎn)可能跨越357-722aa的整個(gè)區(qū)域。初步的蛋白水平研究發(fā)現(xiàn)357-722aa這段區(qū)域參與Abba1介導(dǎo)細(xì)胞產(chǎn)生ruffling與GTP-Rac1蛋白水平的下調(diào)有關(guān)。
[Abstract]:BACKGROUND & OBJECTIVE : Cell motility is an important pathological and physiological basis in human life activities . It is closely related to aging , angiogenesis and tumor metastasis . The membrane structure remodeling is an important structural foundation of cell movement . It is considered that the protein family of IMD ( IRSp53 / MIM homology domain ) is involved in the remodeling of cell membrane structure . In the present study , the family member is Abba1 . In vitro experiments , Abba1 and Abba1 - IMD are transfected into cells . Methods : The whole length of hAbba1 ( human Abba1 , human Abba1 ) or hAbba1 sequence fragment was loaded into the expression vector of GFP green fluorescent protein , and the recombinant expression plasmid of Abba1 was formed , and the recombinant expression plasmid of Abba1 was formed . It includes 4177 ( - hAbba1 - del - WH2 ) 4178 ( HAbba1 - del - WH2 ) 4178 ( HIRSp53 - IMD - hAbba1 - SRD1 - SRD2 - WH2 ) 4842 ( - hAbba1 - IMD - hAbba1 - SRD1 - SRD2 - WH2 ) 5231 ( - hAbba1 - del - 357 - 553aa - WH2 ) 5232 ( - hAbba1 - del - 357 - 722aa - WH2 ) 5399 ( - hAbba1 - del - 554 - 722aa - WH2 ) . The recombinant expression plasmid of the above - mentioned Abba1 gene was verified by double digestion and gene sequencing . The recombinant expression plasmids were transiently transfected into COS7 cells , and GFP green fluorescent protein was stained by immunofluorescence to show the successful COS7 cells , and the changes of cell membrane structure were observed under the microscope . Results : The results showed that , compared with the GFP control group , the total length Abba1 promoted the formation of ruminal , and the gene recombinant expression plasmid with similar membrane bulge structure with the transfected full - length Abba1 did not form ruminal , but rather the COS7 cells transfected with the transfected full - length Abba1 did not form ruminal , but it was more specific that the COS7 cells transfected with 4178 ( HAbba1 - IMD - ( Lys 13Glu13 ) - SRD1 - SRD2 - WH2 ) did not form ruminal , but the results of similar results also occurred in COS7 cells expressing the recombinant expression plasmid 4842 ( antisense - hAbba1 - IMD - hIRSp53 - SH3 - WH2 - like ) and 5232 ( - hAbba1 - del - 357 - 722aa - WH2 ) . It was found that the level of GTP - Rac1 protein in COS7 cells expressing full - length Abba1 increased ( P0.05 ) , and the level of GTP - Rac1 protein in COS7 cells expressing 4178 and 5232 was significantly lower than that of control group ( P 0.05 ) . Conclusion : Abba1 alone IMD mainly promotes the formation of microvesicles . In the full - length Abba1 sequence , IMD is involved in regulating the production of ruminal and 13 lysine as the main functional sites . Therefore , IMD is the important functional area for regulating the remodeling of different cell membrane structures . The results suggest that the region is involved in the formation of rumination by Abba1 . The preliminary protein level study found that the region involved in Abba1 - mediated cell production ruminal is related to the downregulation of the level of GTP - Rac1 protein .

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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本文編號：1425110