富含半胱氨酸的酸性分泌糖蛋白在体外调节人平滑肌细胞动脉稳态的实验研究

发布时间：2019-06-04 20:10

【摘要】：目的:颅内动脉瘤是一个威胁生命的疾病。其是自发性蛛网膜下腔出血的首要病因,一旦动脉瘤发生破裂出血,有极高的致残率和死亡率。颅内动脉瘤的确切发病机制仍不十分明确,并且缺乏对颅内动脉瘤的发生进行筛查和对其破裂的风险进行评估特异的生物学标志物。通过文献回顾,本课题组提出"动脉稳态"(Arterial Homeostasis)的概念,即对于血流应力和血管退行性变所造成的损伤,动脉血管自身存在着一定修复的能力,从而形成损伤与修复的动态平衡来维持血管稳定,这种平衡的打破可能是颅内动脉瘤的发病机制。遗传学因素在颅内动脉瘤发病过程中所起的作用日益受到人们的关注。研究表明,富含半胱氨酸的酸性分泌糖蛋白(secreted protein acidic and rich in cysteine,SPARC)广泛地表达于颅内动脉瘤组织平滑肌细胞层,而其对平滑肌细胞的作用尚不十分清楚。本论文旨在研究SPARC对人血管平滑肌细胞的作用,以及这种作用是否影响了动脉稳态,从而探讨其在颅内动脉瘤发生、发展过程中可能起到的作用。方法:本研究采用原代人脐动脉血管平滑肌细胞(human umbilical arterial smooth muscle cells,HUASMCs)作为研究对象,将其分别暴露于含有(0、0.125、0.25、0.5、1、2和4ug/ml)SPARC的培养基培养24小时后,采用细胞计数试剂盒(CCK8)检测HUASMCs的细胞活力。将其分别暴露于含有(0、2ug/ml)SPARC的培养基2、6、12、24和48小时后,采用流式细胞术检测HUASMCs的细胞周期分布和细胞凋亡比率的变化;并用Western Blot技术检测HUASMCs中以下蛋白表达情况:细胞周期调控蛋白(P21、P53、CyclinD1),细胞凋亡相关蛋白(Bax、Bcl2、Caspase-3、Cl eaved Caspase-3),明胶酶和金属蛋白酶组织抑制剂(MMP2、MMP9、TIMP2、TIMP1)。结果:将HUASMCs暴露于含有(2、4ug/ml)SPARC的培养基,作用24小时后,细胞活力相较对照组分别为89.30±2.00%和87.57±2.17%(P0.05 vs.对照组)。暴露于含2ug/mlSPARC的培养基,作用12小时后,处于细胞周期G0/G1期的细胞比例为74.77±1.33%(P0.05vs.对照组);作用24小时后,处于早期凋亡和晚期凋亡的细胞比例分别为7.38±1.25%和4.86±0.81%(P0.01 vs.对照组)。将HUASMCs暴露于含有2ug/ml SPARC的培养基,作用24小时后,蛋白免疫印迹灰度分析结果显示,P21蛋白含量在2-12小时显著提高(P0.05 vs.对照组),而P53蛋白的表达量在48小时内并没有显著地改变(P0.05 vs.对照组);同时,Bax蛋白水平显著地上调并于24小时达到高峰(P0.01 vs.对照组),而Bcl2蛋白在24小时内无显著变化但在48小时出现显著地下调(P0.01 vs.对照组),Cleaved caspiase3蛋白也显著上调并在24小时达到高峰(P0.05 vs.对照组);MMP2蛋白的含量显著增加且在24小时达到高峰(P0.01 vs.对照组),TIMP2蛋白的表达水平相对保持稳定;MMP9蛋白的表达量显著增加,同时也伴随着TIMP1蛋白含量的显著上调(P0.05vs.对照组)。实验结果提示,SPARC可以通过提高P21蛋白的表达将HUASCMs细胞阻滞于细胞周期的G0/G1期,抑制HUASMCs细胞的增殖速度,还可以促进HUASMCs进行线粒体途径的细胞凋亡,从而降低其细胞活力;SPARC也可能通过促进明胶酶(MMP2、MMP9)的分泌,导致血管壁中细胞外基质和内弹力层的退行性变。结论:实验结果提示,SPARC可能通过增加对颅内动脉中膜、内弹力层及其他细胞外基质的损伤因素,同时降低其自我修复能力,打破动脉血管损伤与修复的动态平衡,导致颅内动脉瘤的发生、发展、甚至破裂。
[Abstract]:Objective: The intracranial aneurysm is a life-threatening disease. It is the primary cause of the spontaneous subarachnoid hemorrhage, and once the aneurysm ruptures, there is a very high rate of disability and mortality. The exact pathogenesis of the intracranial aneurysm is still not very clear, and the lack of screening for the occurrence of intracranial aneurysms and the assessment of specific biological markers for the risk of their rupture. According to the literature review, the research group put forward the concept of the "arterial steady state" Homeostat, that is, to the damage caused by the blood flow stress and the degeneration of the blood vessel, the arterial blood tube has the ability to repair itself, so that the dynamic balance of the injury and the repair is formed to maintain the blood vessel stability, The break in this balance may be the pathogenesis of an intracranial aneurysm. Genetic factors play an increasingly important role in the pathogenesis of intracranial aneurysms. It is shown that the acidic secreted glycoprotein (SPARC), which is rich in cysteine, is widely expressed in the smooth muscle cell layer of the intracranial aneurysm, and its effect on smooth muscle cells is not clear. The purpose of this paper is to study the effect of SPARC on the human vascular smooth muscle cells, and whether the effect of this effect on the steady state of the artery, thus to explore the possible role of SPARC in the occurrence and development of the intracranial aneurysm. Methods: The primary human umbilical artery smooth muscle cells (HUASMCs) were used as the research object, and the cells were exposed to a medium containing (0, 0.125, 0.25, 0.5,1,2 and 4 ug/ ml) for 24 hours, and the cell viability of HUASMCs was detected by a cell count kit (CCK8). The cell cycle distribution and cell apoptosis rate of HUASMCs were detected by flow cytometry after 2,6,12,24 and 48 hours of medium containing (0,2 ug/ ml) of SPARC, respectively. The expression of the following proteins in HUASMCs was detected by Western Blot technique: cell cycle control protein (P21, P53, CyclinD1), Apoptosis-related proteins (Bax, Bcl2, Caspase-3, Ceaved Caspase-3), gelatinase and metalloprotease inhibitor (MMP2, MMP9, TIMP2, TIMP1). Results: HUASMCs were exposed to a medium containing (2,4 ug/ ml) of SPARC, and after 24 hours, the cell viability was 89.30% and 87.57% 2.17%, respectively (P0.05 vs. control group). After 12 hours of exposure to a medium containing 2 ug/ ml of SPARC, the proportion of cells in the G0/ G1 phase of the cell cycle was 74.77 and 1.33% (P0.05 vs. The proportion of cells in early and late-stage apoptosis was 7.38-1.25% and 4.86-0.81%, respectively (P0.01 vs. control group). When HUASMCs were exposed to a medium containing 2 ug/ ml of SPARC for 24 hours, the results showed that the content of P21 protein increased significantly in 2-12 hours (P0.05 vs. control group), while the expression of P53 protein did not change significantly within 48 hours (P0.05 vs. control group); at the same time, The level of Bax protein was up-regulated and peaked at 24 h (P0.01 vs. control group), while the Bcl2 protein did not change significantly within 24 hours, but decreased significantly in 48 hours (P0.01 vs. control group), and Cleared casei 3 protein was also up-regulated and reached its peak at 24 h (P0.05 vs. control group); The content of MMP2 protein was significantly increased and reached the peak at 24 h (P0.01 vs. control group), and the expression level of TIMP2 protein was relatively stable; the expression of MMP9 protein was significantly increased, with a significant increase in the content of TIMP1 protein (P0.05 vs. Control group). The results suggest that SPARC can block the proliferation of HUASMCs by increasing the expression of P21 protein in the G0/ G1 phase of the cell cycle, inhibit the proliferation of the HUASMCs, and promote the apoptosis of the cells of the HUASMCs in the mitochondria pathway, thereby reducing the cell viability of the HUASMCs. SPARC may also promote the gelatinase (MMP2, The secretion of MMP9 leads to the degeneration of the extracellular matrix and the inner elastic layer in the vascular wall. Conclusion: The results suggest that SPARC may increase the damage to the media, inner elastic layer and other extracellular matrix of the intracranial artery, reduce its self-healing ability, break the dynamic balance of the arterial injury and repair, and result in the occurrence and development of the intracranial aneurysm. And even break.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743

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