色素上皮衍生因子通过调节Bcl-2的表达参与砷致人肝细胞线粒体凋亡

Pigment epithelium-derived factor may play a role in apoptosis of human liver cells induced by arsenic through regulating Bcl-2 level

目的探讨色素上皮衍生因子（PEDF）在砷诱导人正常肝细胞线粒体凋亡中的作用机制。方法体外培养人正常肝细胞系HHL-5，亚砷酸钠处理24h，分别为O（对照）、2、5、8、10μmol／L组，流式细胞仪检测线粒体膜电位变化，实时荧光定量PCR检测PEDF、B淋巴细胞瘤．2基因（Bcl-2）、B淋巴细胞瘤．2基因相关X蛋白（Bax）mRNA表达水平。结果与对照组（688．67±47．50）比较，10μmol／L组（562．33±31．50）线粒体膜电位显著降低（P〈0．01），5μmol／L组（634．50±49．03）、8μmol／L组（655．67±13．65）未见明显变化（P均〉0．05）。与对照组[（86．69±12．19）％]比较，2μmol／L组PEDFmRNA表达水平[（81．52±13．93）％]未发生明显变化（P〉0．05）；5、8、10μmol／L组PEDFmRNA表达水平[（59．35±11．21）％、（58．46±8．69）％、（51．57±7．58）％]均显著降低（P均〈0．01），其中10μmol／L组PEDFmRNA表达量最低。与对照组[（96．48±31．14）％]比较，10μmol／L组Bcl-2mRNA表达水平[（53．53±21．49）％]明显降低（P〈0．05）；与对照组[（84．47±35．38）％]比较，5、8、10μmol／L组BaxmRNA表达水平[（65．14±32．34）％、（71．81±12．61）％、（72．224-28．40）％]未发生明显变化（P均〉0．05）。结论PEDF可能通过调节Bcl-2表达水平，参与砷致人肝细胞线粒体凋亡。

Objective To explore the mechanism of pigment epithelium-derived factor （PEDF） on human liver cells in mitoehondrial apoptosis induced by arsenic. Methods Human normal liver cell line （HHL-5） was exposed to NaAsO2 for 24 h, the dose was 0 （control）, 2, 5, 8 and 10 μmol/L, respectively. The mitochondrial membrane potential was determined by using flow cytometry, and mRNA levels of PEDF, Bcl-2 and Bax were assayed by Real-time fluorescent quantitative PCR. Results The mitochondrial membrane potential in 10 μmol/L arsenic group （562.33 ± 31.50） was decreased markedly in comparison with that of control group （688.67 ± 47.50, P 〈 0.01）, the levels in other groups （5 μmol/L： 634.50 ± 49.03, 8 μmol/L： 655.67 ± 13.65） were not significantly changed （P 〉 0.05）. Compared with control group [（86.69 ± 12.19）%], the mRNA levels of PEDF decreased significantly （P 〈 0.01） when the HHL-5 cells were cultivated with 5 μmol/L [（59.35 ± 11.21）%], 8 p, mol/L [（58.46 ± 8,69）%] and 10 μ mol/L [（51.57 ± 7.58）%] arsenic, respectively, and the PEDF mRNA level in 10 μmol/L group was the lowest; but there was no significant change in PEDF mRNA level [（81.52 ±13.93）%] when HHL-5 cell was cultivated with 2μmol/L arsenic. Similarly, mRNA level of Bcl-2 in 10μmol/L arsenic group [（53.53 ± 21.49）%] was significantly decreased compared with that of the control group [（96.48 ± 31.14）%, P 〈 0.05]. Whereas, Bax mRNA levels in arsenic exposure groups [（65.14 ± 32.34）%, （71.81 ± 12.61）%, （72.22 ± 28.40）%] were not significantly changed in comparison with that of control group [（84.47 ± 35.38）%, P 〉 0.05]. ConclusionPEDF may play a role in mitochondrial apoptosis of human liver cells induced by arsenic through regulating Bcl-2 level.