一种用流式细胞术检测基因转移效率的新方法

A Method to Detect Gene Transfection Efficacy by Flow Cytometry

背景与目的:基因转移效率一直以来都是用表达报告基因的细胞(显色或发荧光)占细胞总数的百分比(即转染率)来表示,此百分比是由研究者借助显微镜(或荧光显微镜)计数得出来的。该方法(本文称为人工计数法)存在客观性、准确性不够和工作量大、且不能同时检测基因表达效率等问题。本研究拟寻找一种客观、准确、简便地检测基因转移和表达效率的新方法。方法:以绿色荧光蛋白(GFP)基因为目的基因、重组腺病毒(AdCMV/GFP)为基因载体,用人工计数法测定其对肝癌细胞株HepG2的转染率;用流式细胞术(flowcytometry,FCM)测定其对肝癌细胞株HepG2、Bel7402、Hep3B、SMMC7721和鼻咽癌细胞株CNE-2的转染效率和GFP(绿色荧光蛋白)基因的表达效率(以荧光指数表示)。结果:以FCM测得AdCMV/GFP对HepG2的转染率与载体量呈对数相关(与人工法相似,但前者略高),荧光指数与载体量呈线性相关(r=0.9984),其它细胞株的结果与HepG2相似。结论:采用FCM法测定基因转移效率克服了主观因素的影响,测定结果客观准确,可以用于测定基因载体对真核细胞的转染效率,或研究转录调控元件的功能。

Background &Objective:Analysis of gene transfer and expression is conventionally inferred fro m the percentage of positive cells expressing reporter gene in total cells,re ferred as transfection rate,by investigators counting under a microscope or fluoroscope,w hich was called as manual counting.B ut in many cases,it is not accurate an d easily influenced by the subjectivity of ob server.This study was designed to se ek a convenient method to assess obje ctively and accurately the efficacy of gene tran sfer and expression.Methods:Hepatocellular carcinoma (HCC)HepG2cells were infected with a recombinant adenovirus expressing green fluorescent pr otein(AdCMV /GFP)at a series of multiplicities of infection(MOIs).24h later,the transfection rates w ere assessed by manual counting under fluorescent microscope.Meanwhile,besides transfection ra tes,fluorescent indices(FIs)which indicated the efficiency of ge ne transfer and expression were analyzed by flow cytometry(FCM).Transfection efficiencies of AdCM V /GFP to HCC Hep3B,Bel7402,SMMC7721cells and nasopharyngeal carcinoma CNE-2cells were also tested by FCM.Results:Although transfection rates by FCM w ere slightly higher than that by manual c ounting,both were logarithmic corr elative with vector doses.The stirring was that FIs by FCM showed compellent linear correlation with vector doses(r=0.9984,P<0.001).The efficiency of gene transfer in other cells by FCM were similar to that in HepG2.Conclusion:The efficiency of gene transfer and e xpression in mammalian cells can be easily analyzed by flow cytome try,which is more sensitive,objective,and accurate than manual counting,especially in assessing the efficie ncy of multiple gene transfer(multi-copies per cell)and expression.