摘要
目的 探讨Rxa对过氧化物所致肝细胞凋亡时细胞保护作用的可能分子机制。方法应用全细胞膜片钳单细胞逆转录聚合酶链反应 (RT PCR)技术进行Rxa对过氧化氢 (H2 O2 )诱导人类肝细胞系 (L0 2细胞 )FasmRNA表达的单细胞分析 ,应用全细胞膜片钳显微荧光单细胞浆游离钙浓度 ([Ca2 + ]i)测量技术进行同期瞬时Ca2 + 流变化观察。用流式细胞术观察早期凋亡细胞指数。结果 H2 O2 作用于L0 2细胞 2h电泳图分析可见FasmRNA表达 ,[Ca2 + ]i (1115 .2 8± 2 2 7.16 )nmol/L、早期凋亡细胞指数骤升为 16 .18± 0 .6 5 ;而同期Rxa处理组电泳图分析未见FasmRNA特异性扩增条带出现 ;[Ca2 + ]i、早期凋亡细胞指数较低 (P <0 .0 1、P <0 .0 1)。结论 Rxa可能通过抗氧化和钙阻滞作用阻抑L0 2细胞胞浆段Fas信号传导途径的活化 ,发挥细胞保护作用。
Objective To investigate the probable molecular mechanism of cytoprotective effect of Rxa against H 2O 2 mediated hepatocytes apoptosis. Methods Fas mRNA expression in human hepatocyte (L02 cell)was analyzed by whole cell patch clamp combined with single cell reverse transcription polymerase chain reaction (RT PCR), the cytosolic Ca 2+ concentration was measured by using whole cell patch clamp combined with single cell microfluorescence [Ca 2+ ]i measurement, the index of early phase apoptotic cells (Annexin V + cells) measured by using flow cytometry (FCM), and the hepatocytic morphological changes were observed under scanning electron microscopy. Results There was overexpression of Fas mRNA from single L02 cells in H 2O 2 injury group (10 mol/L). An elevated Ca 2+ wave and [Ca 2+ ]i could be recorded by the whole cell patch clamp [(1?115.28±227.16)?nmol/L]. The index of Annexin V + cells was increased significantly (16.18±0.65). By contrast, there was no expression of Fas mRNA from single L02 cells pre treated with Rxa (2 mmol/L) at the same time. The [Ca 2+ ]i and index of Annexin V + cells in this group were lower than in non pretreatment group. Conclusion The cytoprotective effect of Rxa was associated with down regulating expression of Fas mRNA. Probably, it may be correlated with blocking of Ca 2+ influx.
出处
《中华实验外科杂志》
CSCD
北大核心
2000年第2期155-157,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金! ( 3 9770 93 8)
