自噬与MALAT1在ox-LDL介导血管细胞增殖中的作用及机制研究

发布时间：2018-05-06 11:45

  本文选题：内皮祖细胞 + 自噬　； 参考：《第三军医大学》2017年硕士论文

【摘要】：研究背景及目的目前,心血管病死亡占城乡居民总死亡原因的首位,有统计显示农村为44.6%,城市为42.51%。心血管病的疾病负担日渐加重,已成为重大的公共卫生问题。在心血管病中动脉粥样硬化(Atheroslerosis,AS)严重危害人类健康。在AS形成过程中血管内膜的损伤是其始动环节,内皮通透性、黏附性和血液凝固性的改变及所释放的大量细胞因子导致血管壁发生一系列连锁反应,从而导致血管壁结构的改变。具体表现为内膜的脂质浸润、血管平滑肌细胞(vascular smooth muscle cell,VSMC)和巨噬细胞迁移和泡沫细胞的形成其中,VSMC是增殖体系中最活跃的细胞。即内皮细胞的损伤和VSMC的异常增殖是AS发生发展过程中两个重要环节,所以针对内皮损伤的修复及VSMCs异常增殖的防治能促进血管损伤的修复,对于AS的预防和治疗至关重要。而内皮祖细胞(Endothelial Progenitor Cell,EPC)被认为是内皮细胞损伤后修复的关键细胞。所以我们的研究是基于在AS危险因素——氧化低密度脂蛋白(oxide low density lipoprotein,ox-LDL)环境下,分两部分对EPCs和VSMCs增殖能力的变化和相关机制进行探索。第一部分自噬在氧化低密度脂蛋白诱导的EPCs增殖中的作用EPCs在1997年被首次发现并且命名。之后的研究发现,EPCs有改善内皮功能,增加血管新生的作用,并且揭示了高脂血症、高血压等AS相关高危因素与EPCs数量的减少密切相关,EPCs数量的减少还可以作为AS进展的独立危险因素。在动物模型中,如兔的脑缺血模型、大鼠急性肾损伤模型等,基于EPCs移植相关的研究也发现EPCs可以改善缺血造成的损伤。但是移植研究距离临床应用还有较大距离。所以在应激环境下,提高EPCs的生存至关重要。自噬是机体在应激环境下产生的一种以高效节约体内物质为基础的自我保护行为。研究发现自噬可以降低凋亡并且维持细胞活性。2011年,科学杂志的一项研究进一步从机制层面揭示了自噬的抗凋亡作用可以通过上调抗凋亡蛋白和清除促凋亡蛋白实现。目前,自噬在心血管系统的内皮细胞、VSMC、心肌细胞等中都有研究,但是自噬对于EPCs的相关研究还比较少见。所以我们的研究旨在通过ox-LDL刺激EPCs,从而观察自噬对EPCs增殖能力的影响。第二部分长链非编码RNA MALAT1在氧化低密度脂蛋白诱导的VSMCs增殖中的作用及机制AS是引起心脑血管疾病的重要因素,这与VSMCs的增殖密切相关。在AS发生发展中,内皮细胞的损伤作为触发点,之后引起一系列改变,导致炎症因子分泌等,最终,造成管腔狭窄的主要细胞成分是异常增殖的VSMC。此外,动脉壁中膜VSMC的增殖或肥大是高血压血管壁增厚的主要原因。所以,针对针对VSMCs异常增殖的研究对于AS及高血压等疾病的防治至关重要。长链非编码RNA(long noncoding RNA,lnc RNA)MALAT-1又名核富集常染色体转录产物2(Nuclera-enriched autosomaltranscript2,NEAT2)属长链非编码RNA家族的重要成员,最早于2003年在非小细胞肺癌研究中被发现。MALAT1在多种组织中表达。近几年的研究也发现,在高糖、缺氧环境下MALAT1可影响血管内皮细胞的功能。然而MALAT1对于与心血管疾病密切相关的VSMCs的作用目前尚未见报道。因此我们旨在通过ox-LDL刺激VSMCs来研究MALAT1对VSMCs增殖的影响,并探索可能的分子机制。方法第一部分1.1我们首先进行了EPCs的培养、鉴定。分离并研碎大鼠脾脏后,用密度梯度离心后将获得的细胞进行培养,待细胞贴壁后在镜下观察细胞形态,并用Di I-Ac-LDL和FITC-UEA-I双染法进行鉴定。1.2采用同样的方法获得EPCs,并将EPCs接种至E-plate8细胞培养板中,使用不同浓度(0μg/ml、10μg/ml、30μg/ml、60μg/ml、100μg/ml)的ox-LDL处理EPCs,继续培养,使用实时细胞分析技术(real time cellular analysis,RTCA)检测各组细胞的增殖活性。1.3使用同上浓度的ox-LDL处理EPCs后,并在不同时间点(0h、6h、12h、18h、24h)提取各组EPCs总蛋白,使用western blot技术进行分析能够反映自噬水平的p62蛋白的相对表达水平。第二部分2.1我们首先培养了人冠状动脉VSMC,不同浓度(0μg/ml、5μg/ml、10μg/ml、20μg/ml、50μg/ml、100μg/ml)ox-LDL处理细胞,使用CCK8检测VSMC增殖能力的变化。2.2并且在不同浓度ox-LDL处理细胞后提取总RNA,使用实时荧光定量PCR检测不同组间的MALAT1的表达变化。2.3合成人源性小干扰RNA(si-malat1),并转染VSMCs细胞,抑制MALAT1的表达。使用实时荧光定量PCR检测转染效率。2.4使用CCK8检测转染后细胞增殖能力的变化,并且比较si-malalt1组和si-malalt1+ox-LDL组增殖能力的变化。2.5使用western blot技术检测增殖相关蛋白p-AKT/AKT在ox-LDL处理、si-malalt1处理及si-malalt1干扰后加ox-LDL处理后各组的表达。2.6在si-malat1处理细胞后,给予AKT激动剂(SC-79)用CCK8法观察VSMC增殖能力的变化。2.7使用小干扰RNA(si-stim1)处理细胞后,用western blot技术检测p-AKT/AKT的表达,用CCK8检测其对细胞增殖的影响。2.8使用si-malat1处理细胞,提取蛋白,检测STIM1的表达。在共聚焦显微镜下结合TG及Ca Cl2检测钙内流情况。结果第一部分1.1成功分离培养EPCs,培养的细胞延伸似梭形,呈典型的EPCs形态。双染法阳性细胞可达85%。1.2 ox-LDL呈浓度依赖性地抑制EPCs的增殖活性。1.3 ox-LDL呈浓度和时间依赖性地抑制p62的表达。1.4 sh Atg7和3-MA抑制自噬,也可以在ox-LDL环境下进一步抑制EPCs的增殖。第二部分2.1 ox-LDL呈浓度和时间依赖性地促进VSMCs的增殖能力。2.2 ox-LDL呈浓度依赖性地促进MALAT1的表达。2.3敲减MALAT1可逆转ox-LDL对VSMCs的促增殖作用。2.4抑制MALAT1可降低ox-LDL诱导的AKT磷酸化。2.5抑制MALAT1可减少AKT激动剂对VMSCs的促增殖作用。2.6抑制MALAT1可抑制STIM1及钙库操纵性钙内流。2.7抑制STIM1也抑制AKT磷酸化及VSMCs增殖。结论第一部分自噬在氧化低密度脂蛋白诱导的EPCs增殖中的作用1.1 ox-LDL抑制EPCs增殖的同时也促进了其自噬水平。1.2自噬可减轻ox-LDL对EPCs增殖的抑制作用。第二部分长链非编码RNA MALAT1在氧化低密度脂蛋白诱导的VSMCs增殖中的作用及机制2.1 ox-LDL可增加MALAT1的表达,同时也促进了VSMCs的增殖。2.2 MALAT1可通过调节AKT的磷酸化,参与ox-LDL对VSMC的促增殖作用。2.3 MALAT1可以通过影响STIM1而影响钙内流及AKT活性,从而影响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.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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