转染血红素氧合酶-1基因减轻人肝细胞体外缺氧-复氧损伤  

The effects of heme oxygenase-1 transfection on hypoxia-reoxygenation injury in vitro of liver cells

作　　者：汪慧访[1] 秦磊 胡明政[3] 钱海鑫[3] 

机构地区：[1]宁波市第一医院普外科,315010 [2]江苏省临床免疫重点实验室 [3]苏州大学附属第一医院普外科

出　　处：《中华器官移植杂志》2009年第1期14-16,共3页Chinese Journal of Organ Transplantation

基　　金：江苏省自然科学基金（BK2007057）

摘　　要：目的探讨血红素氧合酶1重组腺病毒载体（Ad-HO-1）体外转染人肝细胞的转染效果及其对肝细胞缺氧-复氧损伤的影响。方法取人肝细胞系L-02细胞，滴加低温保存的Ad-HO-1，分别培养24h、48h和72h（24h组、48h组和72h组），并以加入空载体腺病毒共培养的L-02细胞为空白对照。采用逆转录聚合酶链反应法检测各组肝细胞HO-1 mRNA的表达水平；以间接免疫荧光标记法检测各组肝细胞的HO-1表达率；在倒置荧光显微镜下观察转染24h和72h的肝细胞中绿色荧光蛋白（EGFP）的表达。取空白对照组肝细胞（培养48h）和48h组肝细胞，缺氧培养4h后再有氧培养8h，采用四甲基偶氮唑盐法测定两组肝细胞存活率。结果24h组、48h组和72h组HO-1 mRNA表达水平明显高于空白对照组，且随着转染时间的延长，HO-1 mRNA的表达水平逐渐升高。空白对照组HO-1的表达率为2．0％，24h组为29％，48h组为85．6％，72h组为84．6％。基因转染后24h和72h，可以观察到L-02细胞中EGFP的表达。经历缺氧-复氧实验后，空白对照组肝细胞的存活率为（37．7±3．5）％，48h组肝细胞的存活率为（89．4±5．2）％，二者相比较，差异有统计学意义（P〈0．01）。结论Ad-HO-1在体外能有效的转染人肝细胞；与未转染者相比，转染肝细胞的缺氧-复氧损伤程度较轻。Objective To investigate the effects of transfection of heme oxygenase-1 adenovirus vector （Ad-HO-1） in vitro on hypoxia-reoxygenation injury of human liver cells. Methods Human liver cell line L-02 cells were cultured for 24, 48 and 72 h （24 h, 48h and 72-h groups）, after adding cryopreserved Ad-HO-1. And L-02 cells were co-cultured with the adenovirus vector vehicle as controls. The HO-1 mRNA expression level of liver cells in each group was detected by reverse transcription PCR. HO-1 expression was detected by indirect immunofluorescence labeling. An inverted fluorescence microscope was applied to observe enhanced green fluorescent protein （EGFP） expression in liver cells 24 and 72 h after transfection. The liver cells in blank control group （culture for 48 h） and 48-h group were cultured for 4 h under hypoxia, followed by culture for 8 h under oxygen condition. The survival rate of cells in two groups was tested by MTT assay. Results HO-1 mRNA expression in 24-h, 48-h and 72-h groups was significantly higher than in control group, and with the prolongation of the transfection time, HO-1 mRNA expression level was gradually increased. In blank control, 24-h, 48-h, and 72-h groups, HO-1 expression rate was 2.0%, 29%, 85.6%, and 84. 6%, respectively. After hypoxia-reoxygenation culture, survival rate in blank control and 48-h groups was （37. 7± 3.5）% and （89. 4 ± 5.2）% respectively （P〈0. 01）. Conclusion Ad-HO-1 in vitro can be effectively transfected into human liver cells. The hypoxia-reoxygenation injury to liver cells with transfection was milder than that to control cells.

关 键 词：血红素氧化酶(脱环) 腺病毒科 细胞低氧 再灌注损伤 肝细胞 

分 类 号：R686[医药卫生—骨科学]