高同型半胱氨酸血症对主动脉组织Bcl-2基因甲基化影响

Effects of hyperhomocysteinemia on Bcl-2 gene methylation of aorta in rats

目的:探讨高蛋氨酸饮食诱导高同型半胱氨酸血症(Hhcy)对大鼠主动脉组织Bcl-2基因启动子区甲基化水平的影响。方法:健康6周龄雄性Wistar大鼠24只,体重(160±10)g,适应性喂养1周后随机分为对照组和Hhcy组,每组12只。对照组给予AIN-93G配方饲料,Hhcy组饲以含1.7%蛋氨酸的AIN-93G配方饲料。喂养18周后,全自动生化仪测定血浆同型半胱氨酸浓度。HE染色观察大鼠主动脉组织形态学变化,免疫组织化学检测大鼠主动脉组织Bcl-2表达;巢式降落式PCR联合甲基化特异性PCR检测Bcl-2基因启动子区甲基化水平,荧光RT-PCR检测主动脉组织Bcl-2基因mRNA表达。结果:18周蛋氨酸饮食可以诱导大鼠Hhcy;HE染色主动脉组织呈现动脉粥样硬化早期形态学变化,免疫组织化学显示Hhcy组主动脉组织Bcl-2表达增加;Hhcy组主动脉组织Bcl-2基因启动子区甲基化水平低于对照组(P<0.05),Bcl-2基因mRNA表达高于对照组(P<0.05)。结论:Hhcy通过降低Bcl-2甲基化水平,使Bcl-2表达增加,平滑肌细胞凋亡减弱,可能是其参与动脉粥样硬化形成的分子机制之一。

Objective：To investigate the effects of hyperhomocystinemia（Hhcy） induced by ingestion of excess methionine on methylation status of Bcl-2 gene in aorta of rats.Method：Twenty-four healthy six-week-old Wistar male rats,weighing（160±10）g.After being held to adapt the environment for one week,the rats were randomly divided into control group（n=12） and Hhcy group（n=12）.The control group was fed with AIN-93G diet.The Hhcy group was fed with high-methionion diet（AIN-93G diet with 1.7% methionion）.After being fed with the previously described diets for 18 weeks,the homocysteine in the plasma was measured with the IMX assays.The thoracic aorta was harvested for morphology（HE staining） and immunohistochemical analysis.The methylation status of Bcl-2 gene was determined by nest touch-down PCR combined MSP（methylation specific PCR）.Real-time PCR was used to detect mRNA expression of arotic Bcl-2.Result：A high methionine diet for 18 weeks was sufficient to induce hyperhomocystinemia;Compared with the control group,the Hhcy group had early atherosclerosis morphological changes by HE staining and elevating Bcl-2 expression by immunohistochemical analysis in aorta.The methylation status of Bcl-2 in Hhcy group was lower than in control group（P0.05）.The mRNA expression of arotic Bcl-2 in Hhcy group was higher than in control group（P0.05）.Conclusion：Reducing smooth muscle cell apoptosis by decreasing methylation status and elevating expression of Bcl-2 in arota may play an important role in the pathogenesis of Hhcy induced atherosclerosis.