瘦素预先给药对L02肝细胞缺氧复氧时细胞凋亡的影响

Effect of ieptin pretreatment on hypoxia-reoxygenation induced apoptosis in human L02 liver cells

目的探讨瘦素预先给药对L02肝细胞缺氧复氧时细胞凋亡的影响。方法L02肝细胞接种于6孔培养板中，孵育24h后，随机分为6组，每组6孔：对照组（C组）、缺氧复氧组（HR组）和不同浓度瘦素预处理组（L1-4组）。HR组于37℃95％N2-5％CO2培养箱中缺氧12h，然后于37℃95％O2-5％CO2培养箱中复氧12h；L1-4组先分别加入瘦素100、200、400和800μg／L，再进行缺氧复氧。取细胞上清液，采用赖氏法测定谷丙转氨酶（ALT）和谷草转氨酶（AST）的浓度；采用Hoechst33342／PI双染色法测定细胞凋亡情况，计算细胞凋亡率；采用荧光定量PCR法测定BaxmRNA和Bcl-2mRNA的表达。结果与C组比较，HR组和L1-4组AEF和AST的浓度升高，早期凋亡率和晚期凋亡率升高，BaxmRNA和Bcl-2 mRNA表达上调（P〈0．01）；与HR组比较，L1-4组ALT和AST的浓度下降，早期凋亡率降低，L组BoxmRNA表达下调，L2组和L3组Bcl-2mRNA表达上调（P〈0．01）；L1-4组间ALT和AST的浓度、早期凋亡率和晚期凋亡率、BaxmRNA和Bcl-2mRNA表达差异无统计学意义（P〉0．05）。结论 瘦素预先给药可抑制L02肝细胞缺氧复氧时细胞凋亡，其机制与上调肝细胞Bcl-2mRNA的表达，下调BaxmRNA的表达有关。

Objective To investigate the effect of ieptin （LEP） pretreatment on hypoxia-reoxygenation （H/R） induced apoptosis in human L02 liver cells. Methods Human L02 liver cells were obtained from pharmacology laboratory, Zhong-Shan University and cultured in DMEM liquid culture medium in an incubator filled with 5% CO2 at 37℃ . The cells were divided into 6 groups （ n = 6 each） ： group control （group C） ; group hypoxia-reoxygenation （group H/R） ; group Ⅰ -Ⅳ pretreatment with LEP 100, 200, 400 and 800μg/L ＋ H/R. In group H/R and group Ⅰ -Ⅳ L02 cells were exposed to 95% N,-5% CO2 fur 12 h followed by 12 h reoxygenation. In group Ⅰ -Ⅳ the cells were pretreated with LEP 100, 200, 400, 800 μg/L respectively before H/R. At the end of 12 h of reoxygenation, the cells were centrifuged and the supernatant was collected for determination of ALT and AST concentrations. Apoptosis in L02 cells was detected by Hoechst 33342/PI staining. Fluorescent quantitative PCR was used to detect Bax and Bcl-2 mRNA expression. Results - （ 1 ） ALT and AST concentrations were significantly increased after H/R. The increase in ALT and AST concentrations was ameliorated by pretreatment with LEP. （2） The H/R-induced apoptotic changes of the cells were attenuated by pretreatment with LEP. （3） The Bax mRNA and Bcl-2 mRNA expression was significantly increased in group H/R as compared with group C. Leptin pretreatrament signiticantly reduced Bax mRNA expression and increased Bcl-2 mRNA expressiml as compared with group H/R. Conclusion LEP pretreatment can decrease H/R-induced apoptosis in the L02 liver cells by down-regulation of Bax mRNA expression and up-regulation of Bcl-2 mRNA expression.