本文選題：血管平滑肌細(xì)胞　切入點(diǎn)：NIH3T3細(xì)胞　出處：《河北醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:細(xì)胞骨架是真核細(xì)胞中與保持細(xì)胞形態(tài)結(jié)構(gòu)和細(xì)胞運(yùn)動(dòng)有關(guān)的纖維網(wǎng)絡(luò),包括微管、微絲和中間絲。細(xì)胞骨架不僅在維持細(xì)胞形態(tài),承受外力、保持細(xì)胞內(nèi)部結(jié)構(gòu)的有序性方面起重要作用,而且還參與許多重要的生命活動(dòng)。Krüppel樣因子4 (Krüppel-like factor, GKLF/KLF4)是KLFs家族中與胚胎發(fā)育、細(xì)胞分化和癌癥發(fā)生密切相關(guān)的轉(zhuǎn)錄因子。作為真核生物轉(zhuǎn)錄因子,KLF4主要分布在細(xì)胞核,參與調(diào)控細(xì)胞增殖、分化、胚胎發(fā)育等重要的生命過(guò)程,是體細(xì)胞重編程為誘導(dǎo)性多功能干細(xì)胞的重要誘導(dǎo)因子之一。目前對(duì)KLF4功能的研究往往局限在細(xì)胞核,關(guān)于KLF4在胞漿的分布及其功能尚鮮有報(bào)道。本文通過(guò)觀察KLF4與細(xì)胞骨架的相互作用,探討KLF4調(diào)節(jié)細(xì)胞骨架重構(gòu)的分子機(jī)制。 方法:用貼塊法分離、培養(yǎng)大鼠血管平滑肌細(xì)胞,胰蛋白酶消化傳代,取2～5代細(xì)胞進(jìn)行實(shí)驗(yàn)。細(xì)胞免疫熒光染色和Western blot分析觀察KLF4在細(xì)胞不同部位的分布;免疫共沉淀檢查KLF4與hhLIM之間的相互作用;激光共聚焦顯微鏡觀察KLF4與細(xì)胞骨架的相互作用;鬼比環(huán)肽染色分析細(xì)胞骨架的變化。 結(jié)果: 1 PDGF-BB促進(jìn)KLF4核輸出 細(xì)胞免疫熒光染色結(jié)果顯示,在靜止的VSMCs中,KLF4主要在胞核中分布。PDGF-BB刺激VSMCs 60 min后,KLF4在胞質(zhì)中的分布增加,刺激120 min后,KLF4在胞質(zhì)中的分布進(jìn)一步增加。Western blot結(jié)果顯示,隨著PDGF-BB刺激VSMCs的時(shí)間的不斷延長(zhǎng),KLF4在胞質(zhì)中的分布明顯增加,呈時(shí)間依賴性,提示PDGF-BB促進(jìn)KLF4的核輸出。 2 KLF4在胞質(zhì)中與細(xì)胞骨架相互作用 免疫雙熒光分析結(jié)果顯示,在靜止的VSMCs中,FITC標(biāo)記的KLF4主要在胞核分布,與TRITC標(biāo)記的α-actin無(wú)明顯的熒光重疊,說(shuō)明KLF4不與actin相互作用。PDGF-BB刺激VSMCs 60 min后,KLF4開(kāi)始從胞核向胞質(zhì)中轉(zhuǎn)移,并與TRITC標(biāo)記的α-actin熒光相互重合為黃色熒光。免疫共沉淀結(jié)果表明,PDGF-BB處理前,在VSMCs的胞質(zhì)中,KLF4抗體不能與α-actin共沉淀,即沉淀物中沒(méi)有出現(xiàn)明顯的α-actin條帶,在給予PDGF-BB刺激后,沉淀物中出現(xiàn)清晰的α-actin條帶。提示,KLF4出核后在胞質(zhì)中以與α-actin相互締合的方式存在。 3 KLF4參與細(xì)胞骨架重構(gòu) Western blot結(jié)果顯示,用細(xì)胞骨架聚合劑(JPK)處理VSMCs后,KLF4在胞質(zhì)中的分布明顯增加;細(xì)胞松弛素(CB),使KLF4在胞質(zhì)中的分布明顯減少,提示,細(xì)胞骨架的重構(gòu)影響KLF4的亞細(xì)胞定位。 用GFP-KLF4融合蛋白表達(dá)載體轉(zhuǎn)染NIH3T3細(xì)胞后,給予PDGF-BB、CB、佛波酯(PMA)、JPK刺激,觀察其對(duì)細(xì)胞骨架重構(gòu)的影響。免疫熒光分析結(jié)果顯示,靜止期的NIH3T3細(xì)胞中含有大量平行束狀分布的應(yīng)力纖維。用PDGF-BB處理細(xì)胞后,束狀分布的應(yīng)力纖維變少,肌絲排列為網(wǎng)狀結(jié)構(gòu);CB使細(xì)胞中的應(yīng)力纖維消失,微絲解聚,呈彌散狀分布于胞質(zhì)中。PMA使細(xì)胞中的應(yīng)力纖維形成podosome小體。JPK使細(xì)胞應(yīng)力纖維變的更加粗大。與未轉(zhuǎn)染組相比, KLF4轉(zhuǎn)染的細(xì)胞中,經(jīng)PDGF-BB處理后,細(xì)胞束狀分布的應(yīng)力纖維無(wú)明顯減少,肌絲仍呈束狀排列; KLF4轉(zhuǎn)染后再經(jīng)CB處理時(shí),應(yīng)力纖維雖然也有解聚,但仍可見(jiàn)部分束狀排列的纖維,說(shuō)明KLF4對(duì)細(xì)胞骨架的解聚具有一定的抵抗作用;KLF4轉(zhuǎn)染后再經(jīng)JPK處理時(shí),細(xì)胞的應(yīng)力纖維與未轉(zhuǎn)染組相比變的更加粗大;KLF4轉(zhuǎn)染后再經(jīng)PMA處理, podosome小體形成明顯減少,這些結(jié)果提示,KLF4參與了細(xì)胞骨架的重構(gòu)。 4 KLF4通過(guò)與hhLIM相互作用促進(jìn)PDGF-BB介導(dǎo)的細(xì)胞骨架重構(gòu) 上述研究結(jié)果顯示,PDGF-BB可以促進(jìn)KLF4與actin相互作用,但KLF4無(wú)典型的actin結(jié)合結(jié)構(gòu)域,那么KLF4是否通過(guò)與actin結(jié)合蛋白hhLIM相互作用參與細(xì)胞骨架重構(gòu)呢?為明確KLF4是否與hhLIM相互作用,我們檢測(cè)KLF4與hhLIM是否能夠免疫共沉淀。免疫共沉淀分析結(jié)果顯示,PDGF-BB促進(jìn)KLF4與hhLIM相互作用。上述結(jié)果說(shuō)明,KLF4通過(guò)hhLIM與細(xì)胞骨架相互作用并調(diào)節(jié)細(xì)胞骨架的重構(gòu)。 結(jié)論: 1 PDGF-BB誘導(dǎo)KLF4出核,且KLF4在胞質(zhì)中與細(xì)胞骨架相互作用。 2 KLF4參與細(xì)胞骨架的重構(gòu)。 3 KLF4通過(guò)與hhLIM相互作用促進(jìn)PDGF-BB介導(dǎo)的細(xì)胞骨架重構(gòu)。
[Abstract]:Objective: the cytoskeleton is fiber network, cell morphology and cell movement of eukaryotic cells and maintain including microtubules, microfilaments and intermediate filaments. The cytoskeleton is not only in the maintenance of cell shape, the external force plays an important role in maintaining order, the internal structure of the cell, but also participate in many important life activities of.Kr ppel like factor 4 (Kr ppel-like factor, GKLF/KLF4 KLFs) is a family of transcription factors and embryonic development, closely related to the occurrence of cell differentiation and cancer. As eukaryotic transcription factor, KLF4 is mainly distributed in the nucleus, is involved in the regulation of cell proliferation, differentiation, embryonic development and other important life processes, is the reprogramming of somatic cells into one of the important inducing factor induced pluripotent stem cells. At present, researches on the function of KLF4 is limited in the nucleus, on the distribution and function of KLF4 in the cytoplasm is rarely reported in this paper. By observing the interaction between KLF4 and cytoskeleton, the molecular mechanism of KLF4 to regulate the remodeling of cytoskeleton was investigated.
Methods: isolated by explant method and cultured rat vascular smooth muscle cells, trypsin, take the 2~5 generation cells were observed. The distribution of KLF4 in different parts of the cell immunofluorescence staining and Western blot analysis; CO immunoprecipitation examining the interaction between KLF4 and hhLIM; confocal laser interaction and observation of KLF4 cytoskeleton microscope; TRITC phalloidin staining analysis of the cytoskeleton.
Result:
1 PDGF-BB to promote KLF4 nuclear output
Immunofluorescence staining showed that in static VSMCs, KLF4 mainly in the nucleus distribution of.PDGF-BB stimulated VSMCs after 60 min, the distribution of KLF4 in the cytoplasm increased after 120 min stimulation, the distribution of KLF4 in the cytoplasm of the further increase of the.Western blot results showed that with the stimulation of PDGF-BB VSMCs time KLF4, distributed in the cytoplasm increased significantly, which was time dependent, suggesting that PDGF-BB promote KLF4 nuclear export.
2 KLF4 interaction with cytoskeleton in cytoplasm
Double immunofluorescence analysis showed that in static VSMCs, FITC labeled KLF4 mainly in the nucleus distribution, alpha -actin and TRITC markers showed no obvious fluorescence overlap, indicating that KLF4 does not interact with actin.PDGF-BB stimulation of VSMCs 60 min, KLF4 began to shift from the nucleus to the cytoplasm, and alpha -actin fluorescence TRITC markers and the overlap for yellow fluorescence. Co immunoprecipitation results showed that PDGF-BB pretreatment in the cytoplasm of VSMCs, KLF4 and -actin alpha antibodies do not co precipitation, sediment that do not appear in the alpha -actin was given in the band, after PDGF-BB stimulation, the sediment appeared a clear -actin bands. That KLF4 exists in the cytoplasm with alpha -actin mutual Association nuclear.
3 KLF4 involvement in cytoskeleton reconstruction
Western blot results showed that after the treatment of VSMCs with cytoskeletal polymerizer (JPK), the distribution of KLF4 in cytoplasm increased significantly. Cytochalasin (CB) reduced the distribution of KLF4 in cytoplasm, suggesting that the cytoskeletal remodeling affected KLF4 subcellular localization.
The expression vector was transfected into NIH3T3 cells with GFP-KLF4 fusion protein, PDGF-BB, CB, phorbol ester (PMA), JPK stimulation, to observe its effect on cytoskeleton remodeling. Immunofluorescence analysis showed that quiescent NIH3T3 cells contain a large number of parallel bundle distribution of stress fibers. PDGF-BB cells treated with bundle the distribution of stress fibers become less myofilament arranged for network structure; CB cell in stress fibers disappeared, depolymerization of actin filaments, are dispersed in the cytoplasm of.PMA cells in stress fiber formation podosome the cell bodies of.JPK stress of fiber become more bulky. Compared with untransfected group KLF4, transfected cells, after PDGF-BB treatment, cell stress distribution in fiber bundle is significantly reduced, myofilament bundles of KLF4; transfection after CB treatment, stress fibers although there are depolymerization, but still visible part of the fiber bundles, KLF4 depolymerization of the cytoskeleton has a certain role in the resistance; KLF4 after transfection by JPK treatment, cell stress fibers and non transfection group compared to become more bulky; KLF4 after transfection by PMA treatment, podosome body formation was significantly reduced, these results suggest that KLF4 is involved in the remodeling of the cytoskeleton.
4 KLF4 promotes PDGF-BB mediated cytoskeleton remodeling through interaction with hhLIM
The results show that PDGF-BB can promote the KLF4 interaction with actin, but no KLF4 binding domains typical of actin, then KLF4 is combined with actin hhLIM protein interactions involved in cytoskeleton remodeling? In order to clarify whether KLF4 interacts with hhLIM, we detected KLF4 and hhLIM could co immunoprecipitation analysis results. Coimmunoprecipitation showed that PDGF-BB promoted KLF4 interaction with hhLIM. These results indicated that KLF4 reconstruction by hhLIM and cytoskeleton interaction and regulation of the cytoskeleton.
Conclusion:
1 PDGF-BB induced KLF4 nucleus, and KLF4 interacts with cytoskeleton in cytoplasm.
2 KLF4 participates in the remodeling of cytoskeleton.
3 KLF4 promotes the cytoskeleton remodeling mediated by PDGF-BB by interacting with hhLIM.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

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本文編號(hào)：1603756