KLF4参与血管平滑肌细胞细胞骨架重构的分子机制研究

发布时间：2018-03-12 23:16

本文选题：血管平滑肌细胞　切入点：NIH3T3细胞　出处：《河北医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:细胞骨架是真核细胞中与保持细胞形态结构和细胞运动有关的纤维网络,包括微管、微丝和中间丝。细胞骨架不仅在维持细胞形态,承受外力、保持细胞内部结构的有序性方面起重要作用,而且还参与许多重要的生命活动。Krüppel样因子4 (Krüppel-like factor, GKLF/KLF4)是KLFs家族中与胚胎发育、细胞分化和癌症发生密切相关的转录因子。作为真核生物转录因子,KLF4主要分布在细胞核,参与调控细胞增殖、分化、胚胎发育等重要的生命过程,是体细胞重编程为诱导性多功能干细胞的重要诱导因子之一。目前对KLF4功能的研究往往局限在细胞核,关于KLF4在胞浆的分布及其功能尚鲜有报道。本文通过观察KLF4与细胞骨架的相互作用,探讨KLF4调节细胞骨架重构的分子机制。方法:用贴块法分离、培养大鼠血管平滑肌细胞,胰蛋白酶消化传代,取2～5代细胞进行实验。细胞免疫荧光染色和Western blot分析观察KLF4在细胞不同部位的分布;免疫共沉淀检查KLF4与hhLIM之间的相互作用;激光共聚焦显微镜观察KLF4与细胞骨架的相互作用;鬼比环肽染色分析细胞骨架的变化。结果: 1 PDGF-BB促进KLF4核输出细胞免疫荧光染色结果显示,在静止的VSMCs中,KLF4主要在胞核中分布。PDGF-BB刺激VSMCs 60 min后,KLF4在胞质中的分布增加,刺激120 min后,KLF4在胞质中的分布进一步增加。Western blot结果显示,随着PDGF-BB刺激VSMCs的时间的不断延长,KLF4在胞质中的分布明显增加,呈时间依赖性,提示PDGF-BB促进KLF4的核输出。 2 KLF4在胞质中与细胞骨架相互作用免疫双荧光分析结果显示,在静止的VSMCs中,FITC标记的KLF4主要在胞核分布,与TRITC标记的α-actin无明显的荧光重叠,说明KLF4不与actin相互作用。PDGF-BB刺激VSMCs 60 min后,KLF4开始从胞核向胞质中转移,并与TRITC标记的α-actin荧光相互重合为黄色荧光。免疫共沉淀结果表明,PDGF-BB处理前,在VSMCs的胞质中,KLF4抗体不能与α-actin共沉淀,即沉淀物中没有出现明显的α-actin条带,在给予PDGF-BB刺激后,沉淀物中出现清晰的α-actin条带。提示,KLF4出核后在胞质中以与α-actin相互缔合的方式存在。 3 KLF4参与细胞骨架重构 Western blot结果显示,用细胞骨架聚合剂(JPK)处理VSMCs后,KLF4在胞质中的分布明显增加;细胞松弛素(CB),使KLF4在胞质中的分布明显减少,提示,细胞骨架的重构影响KLF4的亚细胞定位。用GFP-KLF4融合蛋白表达载体转染NIH3T3细胞后,给予PDGF-BB、CB、佛波酯(PMA)、JPK刺激,观察其对细胞骨架重构的影响。免疫荧光分析结果显示,静止期的NIH3T3细胞中含有大量平行束状分布的应力纤维。用PDGF-BB处理细胞后,束状分布的应力纤维变少,肌丝排列为网状结构;CB使细胞中的应力纤维消失,微丝解聚,呈弥散状分布于胞质中。PMA使细胞中的应力纤维形成podosome小体。JPK使细胞应力纤维变的更加粗大。与未转染组相比, KLF4转染的细胞中,经PDGF-BB处理后,细胞束状分布的应力纤维无明显减少,肌丝仍呈束状排列; KLF4转染后再经CB处理时,应力纤维虽然也有解聚,但仍可见部分束状排列的纤维,说明KLF4对细胞骨架的解聚具有一定的抵抗作用;KLF4转染后再经JPK处理时,细胞的应力纤维与未转染组相比变的更加粗大;KLF4转染后再经PMA处理, podosome小体形成明显减少,这些结果提示,KLF4参与了细胞骨架的重构。 4 KLF4通过与hhLIM相互作用促进PDGF-BB介导的细胞骨架重构上述研究结果显示,PDGF-BB可以促进KLF4与actin相互作用,但KLF4无典型的actin结合结构域,那么KLF4是否通过与actin结合蛋白hhLIM相互作用参与细胞骨架重构呢?为明确KLF4是否与hhLIM相互作用,我们检测KLF4与hhLIM是否能够免疫共沉淀。免疫共沉淀分析结果显示,PDGF-BB促进KLF4与hhLIM相互作用。上述结果说明,KLF4通过hhLIM与细胞骨架相互作用并调节细胞骨架的重构。结论: 1 PDGF-BB诱导KLF4出核,且KLF4在胞质中与细胞骨架相互作用。 2 KLF4参与细胞骨架的重构。 3 KLF4通过与hhLIM相互作用促进PDGF-BB介导的细胞骨架重构。
[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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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