瘦素预先给药对L02肝细胞缺氧复氧时细胞凋亡的影响

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24 h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(L_(1～4)组).HR组于37℃95%N_2-5%CO_2培养箱中缺氧12 h,然后于37℃95%O_2-5%CO_2培养箱中复氧12 h;L_(1～4)组先分别加入瘦素100、200,400和800 μg/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Hoechst 33342/PI双染色法测定细胞捌亡情况,计算细胞凋亡率;采用荧光定量PCR法测定Bax mRNA和Bcl-2 mRNA的表达.结果 与C组比较,HR组和L_(1～4)组ALT和AST的浓度升高,早期凋亡率和晚期凋亡率升高,Bax mRNA和Bcl-2 mRNA表达上调(P<0.01);与HR组比较,L_(1～4)组ALT和AST的浓度下降,早期凋亡率降低,L_3组Bax mRNA表达下调,L_2组和L_3组Bcl-2 mRNA表达上调(P<0.01);L_(1～4)组间ALT和AST的浓度、早期凋亡率和晚期凋亡率、Bax mRNA和Bcl-2 mRNA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-2 mRNA的表达,下调Bax mRNA的表达有关.

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

Objective To investigate the effect of leptin (LEP) pretreatment on hypoxia-reoxygenation (H/R) induced apoptusis 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% CO_2 at 37℃. The cells were divided into 6 groups ( n = 6 each) : group control (group C) ; grouphypoxia-reoxygenation (group H/R); group Ⅰ-Ⅳ pretreatment with LEP 100, 200, 400 and 800 μg/L + H/R. In group H/R and group Ⅰ-Ⅳ L02 cells were exposed to 95% N_2-5% CO_2 for 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 supematant 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 pretreatmcnt significantly reduced Bax mRNA expression and increased Bcl-2 mRNA expression as compared with group H/R. Conclusion LEP pretreatment can decrease H/R-indtwed apoptosis in the L02 liver cells by down-regulation of Bax mRNA expression and up-regulation of Bcl-2 mRNA expression.