过氧化物诱导LO_2细胞凋亡时FasmRNA表达及Ca^(2+)流变化的单细胞分析

Single cell analysis of H_2O_2 mediated Fas mRNA expression and Ca^(2+) influx in LO_2 cells

目的 探讨过氧化物所致肝细胞凋亡时Fas基因表达与Ca2 + 流变化的相互关系。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H2 O2 诱导人类正常肝细胞LO2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胞浆游离钙浓度 ([Ca2 + ]i)测量技术、流式细胞术、扫描电镜观察同期瞬时Ca2 + 流变化及早期凋亡 (Annexin V+ )细胞指数和细胞膜变化。结果 H2 O2 作用 2h单个LO2 细胞电泳图分析可见Fas1mRNA特异性扩增条带 ;单细胞Ca2 +流测定R值明显增强 (P <0 .0 1,t=2 2 .2 42 4) ,[Ca2 + ]i骤升为 1115nmol/L± 2 2 7nmol/L ,与对照组的149nmol/L± 2 3nmol/L比较差异有显著性意义 (P <0 .0 1,t=13.37) ,Annexin V+ 细胞指数亦明显上升 (P <0 .0 1,t=48.41) ,细胞膜起泡。结论 H2 O2 诱导LO2 细胞凋亡时细胞内Ca2 + 失衡可能导致Fas死亡基因的活化。

Objective To investigate whether apoptotic injury is induced by Fas mRNA expression mediated by H 2O 2 and concerned with intracellular Ca 2+ unbalance at a single cell level. Methods Fas mRNA expression in human hepatocyte (LO 2 cell) was analyzed by whole cell patch clamp combined with single cell reverse transcription polymerase chain reaction (RT PCR). Cytosolic Ca 2+ concentration was measured with another whole cell patch clamp combined with single cell microfluorescence [Ca 2+ ]i measurement at the same time. The index of early phase apoptotic cells (Annexin V + cells) measured with flow cytometry (FCM) and the hepatocytic morphological changes were observed by scanning electron microscopy. Results Fas mRNA of LO 2 cells cultured with H 2O 2 (10 μmol/L) was expressed at 2 h. The single cell [Ca 2+ ]i analyzed by another whole cell patchclamp increased to 1 115 nmol/L±227 nmol/L ( P <0.01, t =13.37); at the same time, an elevated Ca 2+ wave was recorded. The index of Annexin V + cells was as high as 16.18±0.65 ( P <0.01, t =48.41), and the membrane surface protuberances were observed. Conclusions The activation of Fas mRNA, as a major apoptotic factor mediated by H 2O 2 in LO 2 cells, is probably associated with the imbalance of intracellular Ca 2+ .