本文選題：內(nèi)皮祖細(xì)胞 + 自噬　； 參考：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景及目的目前,心血管病死亡占城鄉(xiāng)居民總死亡原因的首位,有統(tǒng)計(jì)顯示農(nóng)村為44.6%,城市為42.51%。心血管病的疾病負(fù)擔(dān)日漸加重,已成為重大的公共衛(wèi)生問題。在心血管病中動脈粥樣硬化(Atheroslerosis,AS)嚴(yán)重危害人類健康。在AS形成過程中血管內(nèi)膜的損傷是其始動環(huán)節(jié),內(nèi)皮通透性、黏附性和血液凝固性的改變及所釋放的大量細(xì)胞因子導(dǎo)致血管壁發(fā)生一系列連鎖反應(yīng),從而導(dǎo)致血管壁結(jié)構(gòu)的改變。具體表現(xiàn)為內(nèi)膜的脂質(zhì)浸潤、血管平滑肌細(xì)胞(vascular smooth muscle cell,VSMC)和巨噬細(xì)胞遷移和泡沫細(xì)胞的形成其中,VSMC是增殖體系中最活躍的細(xì)胞。即內(nèi)皮細(xì)胞的損傷和VSMC的異常增殖是AS發(fā)生發(fā)展過程中兩個(gè)重要環(huán)節(jié),所以針對內(nèi)皮損傷的修復(fù)及VSMCs異常增殖的防治能促進(jìn)血管損傷的修復(fù),對于AS的預(yù)防和治療至關(guān)重要。而內(nèi)皮祖細(xì)胞(Endothelial Progenitor Cell,EPC)被認(rèn)為是內(nèi)皮細(xì)胞損傷后修復(fù)的關(guān)鍵細(xì)胞。所以我們的研究是基于在AS危險(xiǎn)因素——氧化低密度脂蛋白(oxide low density lipoprotein,ox-LDL)環(huán)境下,分兩部分對EPCs和VSMCs增殖能力的變化和相關(guān)機(jī)制進(jìn)行探索。第一部分自噬在氧化低密度脂蛋白誘導(dǎo)的EPCs增殖中的作用EPCs在1997年被首次發(fā)現(xiàn)并且命名。之后的研究發(fā)現(xiàn),EPCs有改善內(nèi)皮功能,增加血管新生的作用,并且揭示了高脂血癥、高血壓等AS相關(guān)高危因素與EPCs數(shù)量的減少密切相關(guān),EPCs數(shù)量的減少還可以作為AS進(jìn)展的獨(dú)立危險(xiǎn)因素。在動物模型中,如兔的腦缺血模型、大鼠急性腎損傷模型等,基于EPCs移植相關(guān)的研究也發(fā)現(xiàn)EPCs可以改善缺血造成的損傷。但是移植研究距離臨床應(yīng)用還有較大距離。所以在應(yīng)激環(huán)境下,提高EPCs的生存至關(guān)重要。自噬是機(jī)體在應(yīng)激環(huán)境下產(chǎn)生的一種以高效節(jié)約體內(nèi)物質(zhì)為基礎(chǔ)的自我保護(hù)行為。研究發(fā)現(xiàn)自噬可以降低凋亡并且維持細(xì)胞活性。2011年,科學(xué)雜志的一項(xiàng)研究進(jìn)一步從機(jī)制層面揭示了自噬的抗凋亡作用可以通過上調(diào)抗凋亡蛋白和清除促凋亡蛋白實(shí)現(xiàn)。目前,自噬在心血管系統(tǒng)的內(nèi)皮細(xì)胞、VSMC、心肌細(xì)胞等中都有研究,但是自噬對于EPCs的相關(guān)研究還比較少見。所以我們的研究旨在通過ox-LDL刺激EPCs,從而觀察自噬對EPCs增殖能力的影響。第二部分長鏈非編碼RNA MALAT1在氧化低密度脂蛋白誘導(dǎo)的VSMCs增殖中的作用及機(jī)制AS是引起心腦血管疾病的重要因素,這與VSMCs的增殖密切相關(guān)。在AS發(fā)生發(fā)展中,內(nèi)皮細(xì)胞的損傷作為觸發(fā)點(diǎn),之后引起一系列改變,導(dǎo)致炎癥因子分泌等,最終,造成管腔狹窄的主要細(xì)胞成分是異常增殖的VSMC。此外,動脈壁中膜VSMC的增殖或肥大是高血壓血管壁增厚的主要原因。所以,針對針對VSMCs異常增殖的研究對于AS及高血壓等疾病的防治至關(guān)重要。長鏈非編碼RNA(long noncoding RNA,lnc RNA)MALAT-1又名核富集常染色體轉(zhuǎn)錄產(chǎn)物2(Nuclera-enriched autosomaltranscript2,NEAT2)屬長鏈非編碼RNA家族的重要成員,最早于2003年在非小細(xì)胞肺癌研究中被發(fā)現(xiàn)。MALAT1在多種組織中表達(dá)。近幾年的研究也發(fā)現(xiàn),在高糖、缺氧環(huán)境下MALAT1可影響血管內(nèi)皮細(xì)胞的功能。然而MALAT1對于與心血管疾病密切相關(guān)的VSMCs的作用目前尚未見報(bào)道。因此我們旨在通過ox-LDL刺激VSMCs來研究MALAT1對VSMCs增殖的影響,并探索可能的分子機(jī)制。方法第一部分1.1我們首先進(jìn)行了EPCs的培養(yǎng)、鑒定。分離并研碎大鼠脾臟后,用密度梯度離心后將獲得的細(xì)胞進(jìn)行培養(yǎng),待細(xì)胞貼壁后在鏡下觀察細(xì)胞形態(tài),并用Di I-Ac-LDL和FITC-UEA-I雙染法進(jìn)行鑒定。1.2采用同樣的方法獲得EPCs,并將EPCs接種至E-plate8細(xì)胞培養(yǎng)板中,使用不同濃度(0μg/ml、10μg/ml、30μg/ml、60μg/ml、100μg/ml)的ox-LDL處理EPCs,繼續(xù)培養(yǎng),使用實(shí)時(shí)細(xì)胞分析技術(shù)(real time cellular analysis,RTCA)檢測各組細(xì)胞的增殖活性。1.3使用同上濃度的ox-LDL處理EPCs后,并在不同時(shí)間點(diǎn)(0h、6h、12h、18h、24h)提取各組EPCs總蛋白,使用western blot技術(shù)進(jìn)行分析能夠反映自噬水平的p62蛋白的相對表達(dá)水平。第二部分2.1我們首先培養(yǎng)了人冠狀動脈VSMC,不同濃度(0μg/ml、5μg/ml、10μg/ml、20μg/ml、50μg/ml、100μg/ml)ox-LDL處理細(xì)胞,使用CCK8檢測VSMC增殖能力的變化。2.2并且在不同濃度ox-LDL處理細(xì)胞后提取總RNA,使用實(shí)時(shí)熒光定量PCR檢測不同組間的MALAT1的表達(dá)變化。2.3合成人源性小干擾RNA(si-malat1),并轉(zhuǎn)染VSMCs細(xì)胞,抑制MALAT1的表達(dá)。使用實(shí)時(shí)熒光定量PCR檢測轉(zhuǎn)染效率。2.4使用CCK8檢測轉(zhuǎn)染后細(xì)胞增殖能力的變化,并且比較si-malalt1組和si-malalt1+ox-LDL組增殖能力的變化。2.5使用western blot技術(shù)檢測增殖相關(guān)蛋白p-AKT/AKT在ox-LDL處理、si-malalt1處理及si-malalt1干擾后加ox-LDL處理后各組的表達(dá)。2.6在si-malat1處理細(xì)胞后,給予AKT激動劑(SC-79)用CCK8法觀察VSMC增殖能力的變化。2.7使用小干擾RNA(si-stim1)處理細(xì)胞后,用western blot技術(shù)檢測p-AKT/AKT的表達(dá),用CCK8檢測其對細(xì)胞增殖的影響。2.8使用si-malat1處理細(xì)胞,提取蛋白,檢測STIM1的表達(dá)。在共聚焦顯微鏡下結(jié)合TG及Ca Cl2檢測鈣內(nèi)流情況。結(jié)果第一部分1.1成功分離培養(yǎng)EPCs,培養(yǎng)的細(xì)胞延伸似梭形,呈典型的EPCs形態(tài)。雙染法陽性細(xì)胞可達(dá)85%。1.2 ox-LDL呈濃度依賴性地抑制EPCs的增殖活性。1.3 ox-LDL呈濃度和時(shí)間依賴性地抑制p62的表達(dá)。1.4 sh Atg7和3-MA抑制自噬,也可以在ox-LDL環(huán)境下進(jìn)一步抑制EPCs的增殖。第二部分2.1 ox-LDL呈濃度和時(shí)間依賴性地促進(jìn)VSMCs的增殖能力。2.2 ox-LDL呈濃度依賴性地促進(jìn)MALAT1的表達(dá)。2.3敲減MALAT1可逆轉(zhuǎn)ox-LDL對VSMCs的促增殖作用。2.4抑制MALAT1可降低ox-LDL誘導(dǎo)的AKT磷酸化。2.5抑制MALAT1可減少AKT激動劑對VMSCs的促增殖作用。2.6抑制MALAT1可抑制STIM1及鈣庫操縱性鈣內(nèi)流。2.7抑制STIM1也抑制AKT磷酸化及VSMCs增殖。結(jié)論第一部分自噬在氧化低密度脂蛋白誘導(dǎo)的EPCs增殖中的作用1.1 ox-LDL抑制EPCs增殖的同時(shí)也促進(jìn)了其自噬水平。1.2自噬可減輕ox-LDL對EPCs增殖的抑制作用。第二部分長鏈非編碼RNA MALAT1在氧化低密度脂蛋白誘導(dǎo)的VSMCs增殖中的作用及機(jī)制2.1 ox-LDL可增加MALAT1的表達(dá),同時(shí)也促進(jìn)了VSMCs的增殖。2.2 MALAT1可通過調(diào)節(jié)AKT的磷酸化,參與ox-LDL對VSMC的促增殖作用。2.3 MALAT1可以通過影響STIM1而影響鈣內(nèi)流及AKT活性,從而影響VSMCs的增殖能力。
[Abstract]:Research background and purpose at present, cardiovascular disease death accounts for the first cause of total death in urban and rural areas. Statistics show that 44.6% in rural areas, and that the burden of 42.51%. cardiovascular disease in the city is increasing, and it has become a major public health problem. In cardiovascular disease, atherosclerosis (Atheroslerosis, AS) seriously endangers human health. In the AS shape, it is in the form of AS. The damage of the vascular intima is the starting link, the permeability of the endothelium, the changes of adhesion and blood coagulation, and the release of a large number of cytokines that cause a series of chain reactions that lead to the changes in the structure of the vascular wall. The specific expression is the lipid infiltration of the intima and the vascular smooth muscle cells (vascular smooth muscle). Cell, VSMC) and macrophage migration and the formation of foam cells, VSMC is the most active cell in the proliferation system. That is, the injury of endothelial cells and the abnormal proliferation of VSMC are two important links in the development of AS. Therefore, the repair of endothelial injury and the prevention and treatment of abnormal VSMCs proliferation can promote the repair of vascular injury, and for AS Prevention and treatment are essential. Endothelial Progenitor Cell (EPC) is considered to be the key cell for repair of endothelial cells. Therefore, our study is based on two parts of the AS risk factor, the oxide low density lipoprotein (ox-LDL), in which EPCs and VSMCs are proliferating. The role of autophagy in EPCs proliferation induced by oxidized low density lipoprotein (EPCs) was first discovered and named in 1997. After the study, it was found that EPCs improved endothelial function, increased the role of angiogenesis, and revealed AS related high risk factors such as hyperlipidemia and hypertension and EPCs The decrease in quantity is closely related, and the decrease in the number of EPCs can also be an independent risk factor for the progress of AS. In animal models, such as the rabbit model of cerebral ischemia, the rat model of acute renal injury, and so on, the study based on EPCs transplantation also found that EPCs can improve the damage caused by ischemia. However, there is a great distance from the transplantation study to the clinical application. So in a stressful environment, improving the survival of EPCs is crucial. Autophagy is a self-protection behavior based on high efficiency in the body under stress environment. The study found autophagy can reduce apoptosis and maintain cell activity for.2011 years. A study in the Journal of science further revealed autophagy from the mechanism level. The anti apoptotic effect can be achieved by up-regulation anti apoptotic proteins and eliminating apoptotic proteins. Autophagy is currently studied in endothelial cells, VSMC, and cardiomyocytes in the cardiovascular system, but autophagy is still rare in the study of EPCs. Therefore, our study aims to stimulate the proliferation of EPCs by stimulating the proliferation of EPCs through ox-LDL. The effect of capacity. Second the role of long chain uncoded RNA MALAT1 in oxidative low density lipoprotein induced VSMCs proliferation and mechanism AS is an important factor causing cardiovascular and cerebrovascular diseases, which is closely related to the proliferation of VSMCs. In the development of AS, the injury of endothelial cells is a touch point, and then causes a series of changes, causing inflammation. In the end, the main cell component that causes the stenosis of the lumen is the abnormal proliferation of VSMC., and the proliferation or hypertrophy of the membrane VSMC in the arterial wall is the main cause of the thickening of the blood vessel wall of hypertension. Therefore, the study of abnormal proliferation of VSMCs is essential for the prevention and treatment of AS and hypertension. Long chain non coding RNA (long noncodi). Ng RNA, LNC RNA) MALAT-1, also known as nuclear enriched autosomal transcriptional product 2 (Nuclera-enriched autosomaltranscript2, NEAT2), is an important member of the long chain uncoded RNA family. Early in 2003, it was found that.MALAT1 was expressed in a variety of tissues in non small cell lung cancer studies. In recent years, studies have also found that in high glucose and hypoxia environment, MALAT1 can be found. The function of vascular endothelial cells is affected. However, the effect of MALAT1 on VSMCs, which is closely related to cardiovascular disease, is not yet reported. Therefore, we aim to study the effect of MALAT1 on the proliferation of VSMCs by stimulating VSMCs by ox-LDL and explore the possible molecular mechanism. Method first part 1.1 we first carried out the culture of EPCs and identified it. After the spleens were separated from the rat, the cells were cultured with density gradient centrifugation, and the cell morphology was observed under the microscope after the cells were adhered to the wall. EPCs was obtained by Di I-Ac-LDL and FITC-UEA-I double staining, and EPCs was inoculated into the E-plate8 fine cell culture plate, using different concentrations (0 Mu g/ml, 10 u g/ml, 3). EPCs, 0 mu g/ml, 60 g/ml, 100 g/ml), continued to be cultured. Using the real-time cell analysis technique (real time cellular analysis, RTCA), the proliferation activity of each cell was detected by the ox-LDL treatment of the same concentration, and the total protein was extracted at different time points. Analysis of the relative expression level of p62 protein that can reflect the level of autophagy. Second part 2.1 we first cultured human coronary artery VSMC, different concentrations (0 mu g/ml, 5 mu g/ml, 10 mu g/ml, 20 mu g/ml, 50, g/ml, 100 mu g/ml) ox-LDL processing cells, using CCK8 to detect VSMC proliferation capacity changes.2.2 and after different concentrations of cells treated cells extraction Total RNA, using real-time fluorescence quantitative PCR to detect the expression changes of MALAT1 between different groups and.2.3 to synthesize human small interference RNA (si-malat1), and transfect VSMCs cells to inhibit the expression of MALAT1. The proliferation ability of lt1+ox-LDL group.2.5 used Western blot technique to detect proliferation related protein p-AKT/AKT in ox-LDL treatment, si-malalt1 treatment and si-malalt1 interference and ox-LDL treatment after ox-LDL treatment, and.2.6 in si-malat1 treated cells. After RNA (si-stim1) treatment of cells, the expression of p-AKT/AKT was detected by Western blot, the effect of CCK8 on cell proliferation was detected by using si-malat1 processing cells, protein was extracted and the expression of STIM1 was detected. Under confocal microscopy, the internal flow of calcium was detected with TG and Ca Cl2. Results the first part 1.1 successfully isolated and cultured. The cells extended like the spindle shape and showed a typical EPCs form. The double staining positive cells could reach 85%.1.2 ox-LDL in a concentration dependent manner, inhibiting the proliferation activity of EPCs.1.3 ox-LDL in concentration and time dependent inhibition of p62 expression, the expression of.1.4 sh Atg7 and 3-MA inhibition of autophagy, and further inhibition of proliferation in the ox-LDL environment. Second part 2.1 DL has a concentration and time dependence to promote the proliferation of VSMCs.2.2 ox-LDL in a concentration dependent manner to promote MALAT1 expression.2.3 knockout MALAT1 can reverse the proliferation of ox-LDL to VSMCs.2.4 inhibition MALAT1 can reduce ox-LDL induced phosphorylation inhibition Inhibition of STIM1 and calcium library manipulative calcium influx of.2.7 inhibition of STIM1 and AKT phosphorylation and VSMCs proliferation. Conclusion part 1 autophagy in the oxidation of EPCs induced proliferation of low density lipoprotein (LDL), 1.1 ox-LDL inhibits EPCs proliferation and promotes autophagy at the level of.1.2 autophagy that reduces the inhibitory effect of ox-LDL on EPCs proliferation. Second The role of the long chain non coding RNA MALAT1 in the proliferation of VSMCs induced by low density lipoprotein (LDL) and mechanism 2.1 ox-LDL can increase the expression of MALAT1, and also promote the proliferation of VSMCs in.2.2 MALAT1 by regulating the phosphorylation of AKT and participating in ox-LDL to promote the proliferation of VSMC. The activity of T affects the proliferation of VSMCs.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R54

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