Cofilin的表达对人肝细胞增殖和抗损伤能力的影响  

The cofilin expression and its cytoprotective effect on human hepatocyte damage

作　　者：陈天宇[1] 余元龙[2] 胡泽民[2] 

机构地区：[1]中山大学附属第三医院肝胆外科,广州市510630 [2]中山大学附属广东省中山市人民医院肝胆外科,528403

出　　处：《实用医学杂志》2007年第9期1282-1284,共3页The Journal of Practical Medicine

摘　　要：目的:构建丝切蛋白(cofilin)真核细胞表达载体,并观察其在人正常肝细胞中的表达以及对细胞增殖和抗损伤能力的影响。方法:利用基因重组技术将cofilin与绿色荧光蛋白融合并构建真核细胞表达质粒,通过脂质体介导法导入正常人肝细胞系。用荧光显微镜观察表达情况。采用3H-TdR掺入DNA法、PCNA免疫组织化学观察肝细胞DNA合成,了解肝细胞增殖能力的变化。用CCl4造成急性肝细胞损伤模型,通过测定细胞存活率、细胞内丙氨酸氨基转移酶(ALT)、K+的漏出量了解肝细胞的抗损伤能力。结果:双酶切鉴定证实cofilin片段已克隆到pEGFP-N3载体的BamH Ⅰ和Sal Ⅰ位点之间,转染人正常肝细胞后,利用荧光显微镜观察可见到绿色荧光蛋白的表达。转导cofilin的肝细胞DNA合成明显增加(转染96h后,3H-TdR掺入量为对照组的4.5倍)。PCNA免疫组化结果显示转导cofilin的肝细胞增殖活性增加。肝细胞抗CCl4损伤的能力明显增强,肝细胞存活率显著提高(83%vs61%,P<0.05)。细胞内ALT、K+的漏出显著降低(分别为35.2U/Lvs 61.3U/L,P<0.01;5.59mmol/Lvs 6.02mmol/L,P<0.01)。结论:基因转染cofilin可在肝细胞中表达并有促细胞分裂活性,而且转染cofilin后的肝细胞抗损伤能力增强。Objectives To construct pEGFP-cofilin expression vector, detect its transient expression in transfected human hepatocytes, and investigate the influence of cofilin expression on the proliferative potential and cytoprotective effects in human hepatocytes. Methods Human cofilin cDNA was ligated to pEGFP vector. The recombinant plasmid was transfected into human hepatocyte line QSG-7701 with liposome. The expression of cofilin protein was observed by fluorescence microscopy. Hepatocytes were collected 24,48,72,and 92 hours after transfection to detect the number of 3H-TdR uptake in DNA. DNA synthesis was observed by using PCNA immunohistochemistry. Acute liver cell damage was induced by carbon tetrachloride. Cytoprotective effect was observed by examining the survival rate of hepatocytes and leakage of intracellular alanine transaminase （ALT） and potassium ions. Results Identification of pEGFP-cofilin by enzyme digestion showed that cofilin fragment had been cloned into BamH Ⅰ and Sal Ⅰ sites of pEGFP-N3-. Expression of green fluorescent protein in transfected hepatocytes was observed under fluorescence microscopy. The 3H-TdR uptake became 4.5 times as much as in the control group 96 hours after transfection. After cofilin transfection, survival rate of the CC14-poisoning hepatocytes significantly increased （83% vs 61%,P 〈 0.05）, and leakage of intracelluar ALT and potassium ions decreased （35.2 U/L vs 61.3 U/L, P〈 0.01 ; 5.59mmol/L vs 6.02 mmol/L, P〈 0.01, respectively）. Conclusion Transfected cofilin is expressed in hepatic cells and has the activity of promoting cell division and protecting hepatic cells against damage.

关 键 词：肝细胞生长因子 肝细胞 基因转染 

分 类 号：R363[医药卫生—病理学]