本文選題：切應(yīng)力 + 血管重建��； 參考：《上海交通大學(xué)》2015年博士論文

【摘要】：血管重建(remodeling)是動脈粥樣硬化和高血壓等心血管疾病的發(fā)病基礎(chǔ),機(jī)械應(yīng)力在其中起著重要的調(diào)控作用。血管內(nèi)皮細(xì)胞(endothelial cells,ECs)和血管平滑肌細(xì)胞(vascular smooth muscle cells,VSMCs)是構(gòu)成血管壁的主要細(xì)胞成分;ECs襯于血管壁內(nèi)表面,直接受到血流經(jīng)過所產(chǎn)生的流體切應(yīng)力的作用;VSMCs與ECs在結(jié)構(gòu)上相鄰,在功能上相互影響。ECs受到流體切應(yīng)力和相鄰的VSMCs的共同作用,其穩(wěn)態(tài)(homeostasis)在血管重建中有著重要意義。力學(xué)因素誘導(dǎo)血管重建的機(jī)制目前并未完全闡明,該機(jī)制的研究對于深入了解心血管疾病的發(fā)病基礎(chǔ)及防治都具有重要意義。本實(shí)驗(yàn)室前期的血管組織差異蛋白質(zhì)組學(xué)結(jié)果提示,核纖層蛋白Lamin A可能是力學(xué)敏感蛋白;因此,本文圍繞核膜蛋白Nesprin2、SUN1和Lamin A展開了不同切應(yīng)力條件下ECs增殖和凋亡變化的機(jī)制研究。本文應(yīng)用平行平板流動腔系統(tǒng),對單獨(dú)培養(yǎng)的大鼠胸主動脈ECs施加15 dyn/cm2的正常切應(yīng)力和5 dyn/cm2的低切應(yīng)力,結(jié)果顯示,低切應(yīng)力明顯抑制了核膜蛋白Nesprin2、SUN1和Lamin A的表達(dá),并明顯促進(jìn)了ECs的增殖和凋亡;ECs單獨(dú)培養(yǎng)、ECs與VSMCs接觸聯(lián)合培養(yǎng)以及ECs與VSMCs非接觸聯(lián)合培養(yǎng)3種不同的培養(yǎng)方式中,VSMCs對ECs核膜蛋白的表達(dá)并無顯著影響。ECs分別轉(zhuǎn)染Nesprin2、SUN1和Lamin A的特異性干擾片段后,ECs的核膜蛋白表達(dá)的減少均導(dǎo)致了ECs增殖和凋亡的異常增加。為了闡明核膜蛋白參與低切應(yīng)力誘導(dǎo)ECs增殖和凋亡的機(jī)制,我們對Nesprin2和Lamin A蛋白進(jìn)行了深入研究,分別構(gòu)建了Nesprin2和Lamin A的過表達(dá)質(zhì)粒,發(fā)現(xiàn)低切應(yīng)力條件下ECs的Nesprin2和Lamin A的過表達(dá)能夠逆轉(zhuǎn)低切應(yīng)力誘導(dǎo)的ECs增殖和凋亡。應(yīng)用轉(zhuǎn)錄因子活性芯片技術(shù),將ECs分別進(jìn)行Nesprin2和Lamin A特異性干擾片段的轉(zhuǎn)染和過表達(dá)質(zhì)粒的轉(zhuǎn)染并檢測。芯片結(jié)果顯示,Nesprin2表達(dá)水平的減少能夠顯著提高轉(zhuǎn)錄因子AP-2和TFIID的活性,而Lamin A表達(dá)水平的降低顯著抑制了轉(zhuǎn)錄因子Stat-1、Stat-3、Stat-5和Stat-6的磷酸化。在靜態(tài)干擾條件下和切應(yīng)力加載條件下,再分別對芯片結(jié)果進(jìn)行了驗(yàn)證。結(jié)果證實(shí)了上述轉(zhuǎn)錄因子在切應(yīng)力調(diào)控Nesprin2和Lamin A變化中的作用。然后,應(yīng)用Ingenuity Pathway Analysis(IPA)軟件預(yù)測,得到了Nesprin2調(diào)控的轉(zhuǎn)錄因子AP-2的靶基因PLA2G16、PITX2、GEM、KISS1、KRT14和TFIID的靶基因FOS、LDLR和Gh,以及Lamin A調(diào)控的轉(zhuǎn)錄因子Stat-1、Stat-3、Stat-5和Stat-6的共同靶基因BCL2L1、CCND2、IRF1、IFNG、IL4和TNF。應(yīng)用熒光定量PCR技術(shù),我們找到了參與影響低切應(yīng)力誘導(dǎo)ECs增殖和凋亡的靶基因,即AP-2的靶基因GEM、TFIID的靶基因FOS和LDLR以及Stat-1、Stat-3、Stat-5和Stat-6的共同靶基因BCL2L1、CCND2、IRF1、IFNG和IL4。綜上所述,在低切應(yīng)力條件下,ECs的核膜蛋白Nesprin2、SUN1和Lamin A的表達(dá)異常下降,其中外核膜蛋白Nesprin2表達(dá)下降后引起其下游轉(zhuǎn)錄因子AP-2和TFIID的表達(dá)上升,AP-2通過正調(diào)控其靶基因GEM,TFIID通過正調(diào)控其靶基因FOS并同時(shí)負(fù)調(diào)控靶基因LDLR,最終誘導(dǎo)ECs的增殖和凋亡。另一方面,低切應(yīng)力抑制核纖層蛋白Lamin A表達(dá),從而下調(diào)了Stat-1、Stat-3、Stat-5和Stat-6的磷酸化,Stat蛋白的磷酸化又通過正調(diào)控靶基因BCL2L1、IRF1、IFNG和IL4,并負(fù)調(diào)控CCND2,最終促進(jìn)了ECs的增殖和凋亡。這些研究結(jié)果對闡明低切應(yīng)力誘導(dǎo)ECs功能異常的力學(xué)生物學(xué)機(jī)制發(fā)揮了重要作用,并為動脈粥樣硬化等心血管疾病的發(fā)病基礎(chǔ)研究提供了新的力學(xué)生物學(xué)思路。
[Abstract]:Vascular reconstruction (remodeling) is the basis of cardiovascular diseases such as atherosclerosis and hypertension. Mechanical stress plays an important regulatory role. Vascular endothelial cells (endothelial cells, ECs) and vascular smooth muscle cells (vascular smooth muscle cells, VSMCs) are the main components of the vascular wall; ECs is lined with vascular wall. The inner surface is directly affected by the fluid shear stress produced by the flow of blood flow; VSMCs and ECs are adjacent to the structure, and the function of the interaction of.ECs to the interaction of the shear stress and the adjacent VSMCs, its steady-state (homeostasis) is of great significance in the reconstruction of blood vessels. The study of this mechanism is of great significance to the deep understanding of the basis and prevention of the pathogenesis of cardiovascular disease. The results of vascular tissue differential proteomics at the early stage of our laboratory suggest that the nuclear fibrinolytic protein Lamin A may be a mechanical sensitive protein; therefore, different shear stresses are expanded around the nuclear membrane protein Nesprin2, SUN1 and Lamin A. Study on the mechanism of ECs proliferation and apoptosis. In this paper, a parallel plate flow cavity system was used to exert 15 dyn/cm2 normal shear stress and 5 dyn/cm2 low shear stress on the isolated rat thoracic aorta, and the results showed that the low shear stress obviously inhibited the expression of the nuclear membrane protein Nesprin2, SUN1 and Lamin A, and obviously promoted ECs. Proliferation and apoptosis, ECs alone culture, ECs and VSMCs contact combined culture, and ECs and VSMCs non contact culture 3 different culture methods, VSMCs has no significant effect on the expression of ECs nuclear membrane protein. After.ECs transfected with Nesprin2, SUN1 and Lamin A, the decrease of the expression of nuclear membrane protein leads to the proliferation of.ECs. In order to elucidate the mechanism of the involvement of nuclear membrane protein in low shear stress induced ECs proliferation and apoptosis, we have conducted in-depth studies on Nesprin2 and Lamin A proteins and constructed Nesprin2 and Lamin A overexpressed plasmids respectively. It is found that the overexpression of ECs Nesprin2 and Lamin A can reverse low shear stress induced by low shear stress. ECs proliferation and apoptosis. Using transcription factor active chip technology, transfection of Nesprin2 and Lamin A specific interference fragments and transfection and detection of overexpressed plasmids respectively. The microchip results show that the decrease of Nesprin2 expression level can significantly increase the activity of AP-2 and TFIID of the transcription factor, and the expression level of Lamin A is decreased significantly. The phosphorylation of the transcription factors Stat-1, Stat-3, Stat-5 and Stat-6 was inhibited. The results of the chip were verified under static and shear stress loading conditions. The results confirmed the role of the above transcriptional factors in the change of Nesprin2 and Lamin A in the shear stress. Then, the Ingenuity Pathway Analysis (IPA) software was used. We found the target genes PLA2G16, PITX2, GEM, KISS1, KRT14 and TFIID, the target genes of the transcriptional factor AP-2, which are regulated by Nesprin2. The target genes that can induce the proliferation and apoptosis of ECs, namely, the target gene GEM of AP-2, the target genes of TFIID, FOS and LDLR, and the common target genes of Stat-1, Stat-3, Stat-5 and Stat-6, are abnormally decreased in the low shear stress conditions, including the outer nuclear membrane protein. When the expression of in2 decreased, the expression of the downstream transcription factor AP-2 and TFIID increased, and AP-2 regulated its target gene GEM, TFIID regulated its target gene FOS and negatively regulated the target gene LDLR, eventually inducing the proliferation and apoptosis of ECs. On the other hand, low shear stress inhibited the A expression of nuclear fibrinolytic Lamin, thus downregulating Stat-1. The phosphorylation of tat-5 and Stat-6, the phosphorylation of Stat protein, also regulates the target genes BCL2L1, IRF1, IFNG and IL4, and negatively regulates CCND2, which ultimately promotes the proliferation and apoptosis of ECs. These results play an important role in elucidating the mechanical biological mechanism of low shear stress induced ECs dysfunction, and for cardiovascular diseases such as atherosclerosis. The basic research of disease provides a new idea of mechanical biology.

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R54

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