【摘要】： 血管平滑肌細(xì)胞(vascular smooth muscle cells,VSMCs)的黏附、增殖與遷移是高血壓、動(dòng)脈粥樣硬化和血管成形術(shù)后再狹窄等血管重塑性疾病的細(xì)胞病理學(xué)基礎(chǔ)。多種生長(zhǎng)因子和細(xì)胞外基質(zhì)(extracellular matrix,ECM),如骨橋蛋白(osteopontin,OPN)、纖連蛋白(fibronectin,FN)等均參與VSMC生物學(xué)行為的調(diào)節(jié)。整合素作為ECM蛋白的受體不僅介導(dǎo)細(xì)胞與ECM之間的相互作用,而且還通過雙向信號(hào)轉(zhuǎn)導(dǎo)功能,控制細(xì)胞黏附、遷移、分化、增殖和凋亡。因而,研究整合素與其ECM配體相互作用所觸發(fā)的信號(hào)轉(zhuǎn)導(dǎo)途徑,以及對(duì)細(xì)胞生物學(xué)行為的影響有助于闡明血管重塑性疾病的分子機(jī)制。 為了闡明OPN與整合素相互作用調(diào)節(jié)VSMC生物學(xué)行為的分子機(jī)制,本研究構(gòu)建整合素β3亞單位胞內(nèi)區(qū)肽段真核表達(dá)載體(pEGFP-C3-β3CD),并人工合成含有β3胞內(nèi)區(qū)不同保守序列(NXXY)的寡肽(肽-747和肽-759),通過導(dǎo)入VSMC,觀察β3亞單位胞內(nèi)區(qū)及其不同區(qū)段在OPN誘導(dǎo)VSMC黏附和遷移功能中所起的作用;并觀察OPN與整合素相互作用對(duì)信號(hào)分子Src、黏著斑激酶(focal adhesion kinase,FAK)、整合素耦聯(lián)激酶(integrin-linked kinase,ILK)的影響,如FAK、ILK的磷酸化水平以及兩者相互作用的變化。在此基礎(chǔ)上,用Src特異性抑制劑PP2、FAK磷酸化特異性抑制劑黏著斑相關(guān)非激酶(focal adhesion related non-kinase,FRNK)、ILK反義RNA或siRNA(ILK siRNAⅠ和Ⅱ),分別阻斷Src、FAK磷酸化或ILK表達(dá),進(jìn)一步探討三者在OPN誘導(dǎo)VSMC黏附和遷移中所起的作用以及三個(gè)信號(hào)分子的上、下游關(guān)系。 1整合素β3亞單位胞內(nèi)區(qū)在OPN誘導(dǎo)VSMC黏附和遷移中的作用 ECM蛋白作為整合素的配體,通過與細(xì)胞膜上的整合素受體結(jié)合而將與整合素β亞單位胞內(nèi)區(qū)相互作用的蛋白募集到胞膜內(nèi)側(cè),共同形成黏著斑(focal adhesion)復(fù)合物,并啟動(dòng)多條信號(hào)轉(zhuǎn)導(dǎo)途徑,從而導(dǎo)致細(xì)胞生物學(xué)行為改變。本部分實(shí)驗(yàn)觀察整合素β3亞單位胞內(nèi)區(qū)及其保守序列
[Abstract]:Adhesion, proliferation and migration of vascular smooth muscle cells (vascular smooth muscle cells,VSMCs) are the cytopathological basis of vascular remodeling diseases such as hypertension, atherosclerosis and restenosis after angioplasty. A variety of growth factors and extracellular matrix (extracellular matrix,ECM), such as osteopontin (osteopontin,OPN) and fibronectin (fibronectin,FN), are involved in the regulation of the biological behavior of VSMC. Integrin, as the receptor of ECM protein, not only mediates the interaction between cells and ECM, but also controls cell adhesion, migration, differentiation, proliferation and apoptosis through two-way signal transduction. Therefore, the study of the signal transduction pathway triggered by the interaction of integrin with its ECM ligands and the effects on cell biological behavior can help to elucidate the molecular mechanism of vascular remodeling diseases. In order to elucidate the molecular mechanism of the interaction between OPN and integrin in regulating the biological behavior of VSMC, an eukaryotic expression vector (pEGFP-C3- 尾 3CD) of integrin 尾 3 subunit was constructed. Oligopeptides (peptide-747 and peptide-759) containing different conserved (NXXY) sequences of 尾 _ 3 intracellular region were synthesized. The role of 尾 _ 3 subunit intracellular region and its different regions in the adhesion and migration of VSMC induced by OPN was observed by introducing VSMC,. The effects of the interaction of OPN and integrin on the signal molecule Src, focal adhesion kinase (focal adhesion kinase,FAK) and integrin coupled kinase (integrin-linked kinase,ILK), such as the phosphorylation level of FAK,ILK and the interaction between them, were observed. On this basis, the phosphorylation or ILK expression of Src,FAK was blocked by (focal adhesion related non-kinase,FRNK), ILK antisense RNA or siRNA (ILK siRNA 鈪，

本文編號(hào)：2369833