肝隔离门静脉注射法肝细胞基因转移的实验研究  

STUDY OF GENE TRANSFERRING VIA PORTAL VEIN TO PREPARE GENE MODIFIED PRIMARY ENGINEERING HEPATOCYTES

作　　者：梁浩晖[1] 王成友[1] 廖允军[1] 夏荣[1] 申群喜[1] 叶秋华[1] 

机构地区：[1]深圳市第二人民医院,深圳518035

出　　处：《肝胆外科杂志》2005年第5期388-390,共3页Journal of Hepatobiliary Surgery

基　　金：深圳市科技计划项目(JH200505130613)

摘　　要：目的以增强型绿色荧光蛋白(enhanced green fluorescen t prote in,EGFP)基因为标记基因,探索门静脉注射法实施肝细胞基因转移并获取基因修饰原代工程肝细胞(gene m od ified prim ary eng ineering hepatocypte,GM PEH)的新方法。方法W istar大鼠70%切肝建立肝再生模型,术后24h肝隔离门静脉注射脂质体-质粒DNA复合物(lipp fectam ine-pEGFP-N 1DNA com p lexes,lipop lexes)转绿色荧光蛋白(green fluorescen t prote in,GFP)基因至肝细胞,对照组注射生理盐水(N S)。转染后15天(分2组,每组5只大鼠:切肝并于门静脉注射lipop lexes组,L;切肝并于门静脉注射N S组,N)均以胶原酶消化、Perco ll液梯离心法获取纯化细胞悬液;以N S组为对照并以GFP为荧光标记物,用流式细胞仪(flow cytom etry,FCM)分析实验组肝细胞的转染率。结果FCM分析L组的平均转染率(%)为3.40±2.09(p<0.05)。结论肝再生模型L ipop lex门静脉途径灌注转染,可获得稳定的EGFP基因表达率。肝隔离门静脉注射法肝细胞基因转移可高效获取基因修饰的原代工程肝细胞。Objective Using enhanced green fluorescent protein gene as a marker to pursuit a new method of gene transferring to hepatocyte via portal vein to prepare gene modified primary engineering hepatocyte（GMPEH）. Methods In vivo transgene and in vitro separating and sorting cells method to prepare GMPEH：Regenerating liver model of Wistar rat was established by partial （70%） hepatectomy and isolated hepatic perfusion （IHP） with liposome-p^EGFP-N1 DNA compund （lipoplexes,Group L;control group with normal saline,NS,Group N）via portal vein was carried out 24 h post-operation. Purified hepatocytes were obtained via collagenase digestion followed by Percoll gradient cnetrifugation 15d post-transfection respectively. The hepatocytes were photographed with fluorescent microscope and the positive cells were screened by flow cytometry（FCM） using Group N as control. Result The mean transfection efficiency（% ） of Group L analyzed by FCM were 3.40% 2.09（p〈0.05）. Conclusion Stable expression efficiency of hepatocytes could be achieved on 15 days post-IHP. Gene transferring to hepatocyte via portal vein is a new feasible method to prepare gene modified primary engineering hepatocytes.

关 键 词：门静脉 基因转移 脂质体 绿色荧光蛋白 

分 类 号：R657.3[医药卫生—外科学]