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目的:研究胱抑素C(cystatin C)对氧化型低密度脂蛋白(ox-LDL)诱导的人血管平滑肌细胞(VSMC)的作用,以探讨cystatin C在心血管疾病中的生物学特性。方法:利用ox-LDL诱导人VSMC,MTT法检测ox-LDL诱导时间及浓度对细胞存活率的影响,确定ox-LDL最佳诱导浓度和时间,将对数生长期的人VSMC随机分成正常细胞组、pCDNA3.1-cystatin C转染组、ox-LDL诱导组以及ox-LDL+pCDNA3.1-cystatin C转染组,检测各组细胞乳酸脱氢酶(LDH)、活性氧(ROS)、三磷酸腺苷(ATP)生成量以及caspase-3活性变化,并通过逆转录聚合酶链反应(RT-PCR)和Western blotting法测定各组凋亡相关基因bax、bcl-2和Bax、Bcl-2蛋白的表达情况。结果:ox-LDL+pCDNA3.1-cystatin C转染组与ox-LDL诱导组比较,LDH、ROS生成量和caspase-3表达量明显降低,差异有统计学意义(P<0.01);ATP生成量显著升高,差异有统计学意义(P<0.01)。RT-PCR和Western blotting法测定显示ox-LDL诱导组促凋亡基因bax及Bax蛋白表达增加、抗凋亡基因bcl-2和Bcl-2蛋白表达减少,ox-LDL+pCDNA3.1-cystatin C转染组Bcl-2表达增加、Bax表达减少,差异有统计学意义(P<0.05)。结论:Cystatin C抑制ox-LDL诱导的VSMC凋亡,其机制可能与其上调Bcl-2及下调Bax表达有关。
Objective: To investigate the effect of cystatin C on oxidized low density lipoprotein (ox-LDL) -induced human vascular smooth muscle cells (VSMCs) in order to investigate the biological characteristics of cystatin C in cardiovascular diseases. Methods: The effects of ox-LDL induction time and concentration on the cell viability were determined by ox-LDL induction. The optimal concentration and time of ox-LDL induction were determined. Human logarithmic growth phase VSMCs were randomly divided into normal cells Group, pCDNA3.1-cystatin C transfection group, ox-LDL induction group and ox-LDL + pCDNA3.1-cystatin C transfection group. The levels of lactate dehydrogenase (LDH), reactive oxygen species (ATP) production and caspase-3 activity. The expressions of bax, bcl-2, Bax and Bcl-2 protein in each group were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting Express the situation. Results: Compared with the ox-LDL induction group, the levels of LDH and ROS and the expression of caspase-3 in ox-LDL + pCDNA3.1-cystatin C transfection group were significantly decreased (P <0.01) The amount was significantly increased, the difference was statistically significant (P <0.01). The results of RT-PCR and Western blotting showed that the expressions of bax and Bax proteins increased, while the expression of anti-apoptotic genes bcl-2 and Bcl-2 decreased. The expressions of ox-LDL + pCDNA3.1-cystatin C The expression of Bcl-2 and Bax decreased in transfection group (P <0.05). CONCLUSION: Cystatin C inhibits the apoptosis of VSMC induced by ox-LDL, which may be related to its up-regulation of Bcl-2 and down-regulation of Bax expression.