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目的:研究葛根素对同型半胱氨酸(homocysteinemia,HCY)诱导的血管内皮细胞凋亡及葡萄糖调节蛋白78(glucoseregulated protein 78,GRP78)基因表达的影响,探讨葛根素抗动脉粥样硬化的机制。方法:建立HCY诱导的人主动脉血管内皮细胞损伤模型,与不同浓度葛根素(10-7mol/L,10-6mol/L,10-5mol/L)共同培养48h,TUNEL法检测细胞凋亡,流式细胞仪测定DNA含量并分析细胞周期,RT-PCR检测GRP78mRNA表达;目的基因克隆、鉴定并测序。结果:荧光显微镜观察显示葛根素组细胞未见明显凋亡形态学改变;葛根素组G0/G1期细胞数明显减少,S期细胞数明显增多,且存在剂量依赖性,与HCY组相比,差异显著(P<0.01);RT-PCR结果显示:葛根素处理组细胞GRP78mRNA表达明显减少,与HCY比较差异显著(P<0.01);测序结果表明,来源于血管内皮细胞的全长648bp的基因片断与兔GRP78基因高度同源。结论:葛根素拮抗HCY诱导的血管内皮细胞凋亡,GRP78基因低表达可能是其抗动脉粥样硬化的作用机制之一。
OBJECTIVE: To study the effect of puerarin on the apoptosis of vascular endothelial cells induced by homocysteinemia (HCY) and the expression of glucose-regulated protein 78 (GRP78), and to explore the mechanism of puerarin against atherosclerosis . Methods: The injury model of human aortic endothelial cells induced by HCY was established and incubated with different concentrations of puerarin (10-7mol / L, 10-6mol / L, 10-5mol / L) for 48h. The apoptosis was detected by TUNEL, The DNA content was determined by flow cytometry and the cell cycle was analyzed. The expression of GRP78 mRNA was detected by RT-PCR. The target gene was cloned, identified and sequenced. Results: Fluorescence microscopy showed no obvious apoptosis morphological changes of puerarin group; puerarin group G0 / G1 phase cells decreased significantly, S phase cells increased significantly, and there is a dose-dependent, compared with the HCY group, (P <0.01). The results of RT-PCR showed that the expression of GRP78 mRNA in puerarin-treated group was significantly decreased compared with HCY (P <0.01). The sequencing results showed that the full-length 648bp gene from vascular endothelial cells The fragment is highly homologous to the rabbit GRP78 gene. CONCLUSION: Puerarin antagonizes HCY-induced apoptosis of vascular endothelial cells, and the low expression of GRP78 may be one of its mechanisms of anti-atherosclerosis.