活体转染离体分离分选法制备基因修饰原代肝细胞的研究  

In vivo transfection and in vitro sorting cells method to prepare gene modified primary engineering hepatocytes:preparation and actualization

作　　者：夏荣[1] 廖允军[1] 王成友[1] 伍建春[1] 唐远志[1] 申群喜[1] 叶秋华[1] 

机构地区：[1]深圳市第二人民医院普外科,广东省深圳518035

出　　处：《中国基层医药》2006年第4期623-625,共3页Chinese Journal of Primary Medicine and Pharmacy

基　　金：广东省深圳市科技计划项目(200405032;JH200505310613A)

摘　　要：目的探索一种高效获取基因修饰原代肝细胞的新方法。方法Wistar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(lipofectamine-pEGFP-N1DNAcomplexes,lipoplex)转绿色荧光蛋白(greenfluorescentprotein,GFP)基因至肝细胞,对照组注射生理盐水(NS)。转染后15d(分为两组,每组5只切肝并于门静脉注射lipoplex组(L组);切肝并于门静脉注射NS组(N组),均以胶原酶消化、Percoll液梯度离心法获取纯化肝细胞悬液;以NS组为对照并以GFP为荧光标记物,用流式细胞仪(flowcytometry,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)%。结论肝再生模型Lipoplex门静脉途径灌注转染,可获得稳定的GFP表达率。活体转染离体分离分选法获取基因修饰原代肝细胞是一种高效,可行的新方法。Objective To investigate a new high-efficiency method of preparation of gene modified primary engineering hepatocytes（GMPEH）. Methods In vivo transfection and in vitro separating and sorting cells（ITISC） method to prepare GMPEH：regenerating liver modet（RLM） of Wistar rat was established by partial（70 % ） hepatectomy and isolated hepatic perfusion （IHP） with liposome- pEGFP- N1 DNA compound （ lipoplexes, group L; Control group with normal saline,NS,group N） via portal vein was carried out 24h postoperation. Purified hepatocytes were obtained via collagenase digestion followed by Percoll gradient centrifugation 15d post-transfection respectively. The hepatocytes were photographed with fluorescent microscopy（FM） and the positive cells were singled out by flow cytometry（FCM） using group N as control. Results The mean transfection efficiency of group L analyzed by FCM was （3.40 ± 2.09） %. Conclusion Stable transfection efficiency of hepatocytes can be achieved on 15 days post-IHP. ITISC is a new feasible method to prepare GMPEH.

关 键 词：肝再生 肝细胞 门静脉 转染 荧光抗体技术 模型 动物 

分 类 号：R575[医药卫生—消化系统]