【作者】 梁江燕；

【导师】 赵占胜；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:血管并发症是糖尿病(diabetes melletus, DM)患者致死的首要原因,动脉粥样硬化(atherosclerosis, AS)与DM共存。DM患者血糖控制不理想与微血管和大血管病变紧密相关,高血糖在DM血管并发症的发展过程中起着主要作用。血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的过量增生是AS的一个主要特征。已证实,细胞增生是内膜增厚的基本特征之一,但现在普遍认为细胞凋亡调节的紊乱也同样重要。AS中VSMCs的大量聚集是细胞高度增生和凋亡减少共同作用的结果。许多研究证实,高浓度葡萄糖可促进AS的发展,增加体外VSMCs的生长率。高糖不仅能显著抑制无血清诱导的体外培养大鼠VSMCs的凋亡,还能增强抗凋亡蛋白Bcl-2和Bcl-xl的表达,此外明显降低人冠状动脉平滑肌细胞的DNA断裂率。过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptorsγ, PPARγ)是核受体超家族的一个转录因子。PPARγ在VSMCs有表达,在机械损伤的动脉壁中表达显著上调。罗格列酮(rosiglitazone, RSG)属于噻唑烷类二酮药物(thiazolidinediones, TZDs),为PPARγ人工合成配体,是临床上用于治疗DM的胰岛素增敏剂。许多资料显示,TZDs还可延缓AS进程。RSG可减少颈总动脉内膜-中层厚度(IMT),而后者是冠状AS的预测指标。RSG还可减少DM小鼠的动脉粥样斑块总面积并抑制斑块中巨噬细胞的聚集。在离体实验中,RSG可抑制人VCSMs增殖并促进其凋亡,但对于高糖诱导的VCSMs增殖、凋亡及其相关分子机制的影响鲜有报道。本实验通过原代培养大鼠胸主动脉VSMCs,研究RSG对高浓度葡萄糖孵育下的VSMCs增殖、凋亡以及Bcl-2、Bcl-xl的影响,探讨RSG对AS的作用机制。方法:1运用改良组织贴块法进行大鼠胸主动脉VSMCs原代培养并传代,应用自然纯化方法纯化细胞,根据细胞形态学特点和α-actin免疫细胞化学染色进行细胞鉴定;2取处于对数生长期的第5~6代细胞,用MTT法检测不同浓度RSG对高糖孵育下细胞增殖活性的影响;3取处于对数生长期的第5~6代细胞,流式细胞术检测RSG对高糖孵育下细胞周期、凋亡率及Bcl-2、Bcl-xl蛋白表达的影响;4取处于对数生长期的第5~6代细胞,Western blot技术检测RSG对高糖孵育下细胞Bcl-2、Bcl-xl蛋白表达的影响。结果:1原代培养时,约80%的组织块接种成活,培养细胞呈典型的“谷峰状”生长,经平滑肌细胞特异的α-actin免疫化学染色后,培养细胞的胞浆着色呈阳性反应;2用MTT法检测VSMCs的增殖活性,发现在高浓度葡萄糖诱导下,VSMCs显著增殖(P<0.05),而高渗组细胞的增殖能力没有明显改变; RSG(30、100μmol/L)可抑制高浓度葡萄糖诱导的VSMCs的增殖(P<0.05),且呈剂量依赖性;用RSG拮抗剂GW9662(10μmol/L)预处理后,RSG的抑制作用部分被拮抗;3用流式细胞仪检测细胞各周期的时相分布,显示高浓度葡萄糖可诱导G0/G1期细胞可向S期转化,而高渗组对细胞各周期均没有影响;RSG可抑制高糖诱导的G0/G1→S,G0/G1期细胞数目明显增多,S期细胞数目减少(P<0.05);GW9662可部分逆转RSG的抑制作用;4用流式细胞仪检测凋亡率及Bcl-2、Bcl-xl蛋白表达量,发现高浓度葡萄糖能显著降低VSMCs的凋亡率,Bcl-2和Bcl-xl蛋白表达均增强(P<0.05),而高渗组对细胞凋亡率及Bcl-2、Bcl-xl蛋白表达量均无影响;RSG(30、100μmol/L)能够促进高糖组VSMCs的凋亡,Bcl-2、Bcl-xl蛋白表达量显著减少,且呈浓度依赖性;拮抗剂GW9662能部分拮抗RSG的促凋亡作用;5 Western blot技术检测RSG对VSMCs Bcl-2、Bcl-xl蛋白表达量的影响,结果显示高浓度葡萄糖能使bcl-xl蛋白的表达明显增强,且糖浓度越高蛋白表达越强,而高渗组对Bcl-xl蛋白表达没有影响;RSG(30、100μmol/L)能显著降低高糖组Bcl-xl蛋白的表达量,且呈浓度依赖性;用GW9662预处理后,Bcl-xl蛋白的表达量又显著上升。Bcl-2蛋白未测出。结论:1高浓度葡萄糖能促进VSMCs增殖,显著降低VSMCs的凋亡凋亡率,增强Bcl-xl、Bcl-2的蛋白表达量;而高渗组对细胞没有影响。提示高糖状态可能是糖尿病动脉粥样硬化发生机制之一。2 30～100μmol/L的RSG均能在体外有效抑制高糖孵育的VSMCs由G0/G1期向S期的转变,降低高糖组VSMCs内抗凋亡蛋白Bcl-xl、Bcl-2的表达,促进其凋亡,呈剂量依赖性。提示RSG可能通过对高糖诱导的VSMCs增殖和凋亡机制的干预,对T2DM血管病变起到了保护作用。更多还原

【Abstract】 Objective: Vascular complications are the leading cause of death in diabetic patients, atherosclerosis is accelerated by the coexistence of diabetes mellitus. Poor control of diabetes is associated with the development of micro- and macroangiopathy, hyperglycemia in diabetic patients is believed to play a major role in the development of these vascular complications. Increased proliferation of vascular smooth muscle cells (VSMCs) is a key feature in the atherosclerotic lesion. It is well established that cell growth is a fundamental feature of intimal hyperplasia, and it is widely accepted that perturbations in the regulation of apoptosis are equally important. Excessive accumulation of VSMCs in atherosclerosis suggests reduced apoptosis and excessive cell proliferation in the lesions. Several findings support the concept that hyperglycemia accelerates the development of atherosclerosis. High concentration of glucose enhances growth rate in cultured VSMCs. Treatment with a high glucose not only significantly attenuate apoptosis in response to serum withdrawal in cultured rat VSMCs, but also markedly increased the expression of Bcl-2 and Bcl-xl. Another reports shows that there was a significant decrease in the DNA fragmentation ratio of human coronary artery smooth muscle cells cultured at high glucose concentration. Peroxisome proliferator-activated receptor (PPARγ) is a transcription factor belonging to the nuclear hormone receptor gene super-family. PPARγis expressed in VSMCs and prominently upregulated in response to mechanical injury of the arterial wall. Thiazolidinediones (TZDs) are synthetic ligands for PPARγand are commonly used as insulin-sensitizing agents in the treatment of type 2 diabetes. Data suggest that TZDs may also retard atherosclerotic disease progression. Treatment with rosiglitazone (RSG) decreased mean common carotid artery (CCA) intima-media thickness (IMT) progression, a surrogate index of atherosclerotic disease progression. Rosiglitazone attenuated total plaque area in diabetic mice and suppressd macrophage accumulation in diabetic aortic plaques. Although some reports indicated that RSG can inhibit cell proliferation and promote apoptosis in human VSMCs in vitro, little is known about the effect of rosiglitazone on proliferation and apoptosis and its related mechanisms of VSMCs treated with high glucose. To elucidate the precise mechanisms of RSG on AS, the effect of RSG on VSMCs proliferation, apoptosis and expression of Bcl-2, Bcl-xl in rat thoractic aorta smooth muscle cells were examined.Methods: 1 The primary and transfer culture were done by modified tissue-piece inoculation and trypsin digestion respectively. The cells were purified by natural passage transfer. The cultured cells were identified by morphological characteristics and immunocytochemistry with antibody toα-actin.2 The fifth to sixth passage purified cells were harvested for the following experiment. The effect of RSG on the cell proliferating viability in high concentration glucose was observed by MTT assay.3 The fifth to sixth passage purified cells were harvested for the following experiment. Flow cytometry was preformed to analyze the influences of RSG on cell cycle distribution, apoptosis rate and Bcl-2, Bcl-xl expression in high concentration glucose.4 The fifth to sixth passage purified cells were harvested for the following experiment. The effect of RSG on Bcl-2, Bcl-xl expression in VSMCs incubated in high concentration glucose was detected by Western blot.Results: 1 Approximately 80% inoculated tissue pieces survived in primary culture. The cultured cells possessed“peak and valley”characteristics for VSMCs Immunocytochemical staining with specific antibody against SM-α-actin demonstrated these cells were positive.2 MTT assays were used to characterize the viability of VSMCs. The proliferating viability of VSMCs induced by high concentration glucose was remarkably increased when compared with the normal glucose group (P<0.05) , and treatment of the cells with mannitol, used as an osmotic control, had no significant effect on apoptosis in rat VSMCs; RSG(30、100μmol/L)can inhibited the proliferation of VSMCs by high glucose treatment (P<0.05), and displayed in concention-dependent; the effects of RSG was attenuated partly after pretreated with GW9662 (10μmol/L).3 To detect the cell cycle progression of VSMCs, flow cytometric analysis was performed. High glucose can induce cell cycle progression from G0/G1 phase to S phase, and the cell cycle progression was not altered by mannitol treatment compared with the control; RSG (30～100μmol/L) pretreated for 48h can inhibit VSMCs cell cycle progression from G0/G1 phase to S phase, the cell number significantly decreased in G0/G1 phase and increased in S phase(P<0.05); GW9662 can suppress the function of RSG.4 We used flow cytometric analysis to determine the apoptosis rate and Bcl-2, Bcl-xl expression in VSMCs. Treatment with a high glucose significantly attenuated apoptosis rate and increased expression of Bcl-2 and Bcl-xl protein in cultured rat VSMCs when compared with the normal glucose group(P<0.05), but the mannitol have no effect on apoptosis rate and expression of Bcl-2 and Bcl-xl protein; Treatment with RSG(30、100μmol/L) can promoted apoptosis of VSMCs and reduced expression of Bcl-2 and Bcl-xl protein in high glucose group in a dose-dependent manner; the effect of proapoptosis can be suppressed partly by GW9662. 5 Western blot demonstrated that high glucose upregulated Bcl-xL protein and depended in glucose concentration, and expression of Bcl-xl protein had no change when treated with mannitol; the expression of Bcl-xl protein induced by high glucose was decreased by RSG (30、100μmol/L) and increased when pretreated with GW9662. However, Bcl-2 was not detected in VSMCs.Conclusions: 1 High glucose can induce the proliferation and inhibit apoptosis by increasing the expression of Bcl-xl and Bcl-2 protein in rat VSMCs; treatment with mannitol had no effect on VSMCs, indicating that high glucose condition may represent one of the mechanisms for atherosclerosis observed in T2DM.2 RSG (30～100μmol/L) can inhibit VSMCs cell cycle progression from G0/G1 phase to S phase, decrease expression of Bcl-2, Bcl-xl protein and promote VSMCs to apoptosis induced by high glucose in cultured VSMCs. These finding suggested that RSG may play a protective role against diabetic angiopathy through the intervention of proliferation and apoptosis in VSMCs.更多还原