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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 possible molecular mechanism of Rxa on cell apoptosis induced by peroxide during hepatocyte apoptosis. Methods Whole cell patch-clamp single-cell reverse transcription-polymerase chain reaction (RT-PCR) was used to perform single cell analysis of Rxa on the expression of Fas mRNA in human hepatocyte cell line (L0 2) induced by H2O2. Whole cell patch Clamp microscopic fluorescent single cell plasma free calcium concentration ([Ca2 +] i) measurement technique for simultaneous transient Ca2 + flow changes were observed. The apoptotic cell index was observed by flow cytometry. Results The expression of Fas mRNA in L02 cells treated with H2O2 for 2 h showed that the expression of FasmRNA was significantly increased in [Ca2 +] i (1115.282 ± 21.76) nmol / L, and the apoptotic index in early stage increased sharply to 16.18 ± 0.6 5; however, there was no FasmRNA-specific amplification band in the Rxa-treated group. The expression of [Ca2 +] i was lower in the early apoptotic cells (P <0.01, P <0.01). Conclusion Rxa may inhibit the activation of Fas signaling pathway in the cytoplasm of L0 2 cells through anti-oxidative and calcium blockade and play a cytoprotective role.