摘要
目的探讨质粒型和腺相关病毒两种转基因载体对家兔关节软骨细胞中GAPDH、B2M、18S rRNA和TUBB内参基因mRNA转录水平的影响。方法将携带增强型绿色荧光蛋白(Enhanced green fluorescent protein,EFGP)报告基因的质粒型载体pcDNA6.2-EGFP和腺相关病毒载体AAV2-EGFP分别转染至体外培养的家兔关节软骨细胞中,荧光显微镜观测转染后关节软骨细胞中EGFP的表达,实时PCR技术检测两各组关节软骨细胞中GAPDH、B2M、18S rRNA和TUBB基因mRNA转录水平的变化。结果转染48 h后,两组转基因关节软骨细胞中均可见明显的绿色荧光;与正常对照组相比,4个内参基因在两组转基因细胞内的转录水平均下调,其中GAPDH和TUBB基因下调较小,腺相关病毒载体对转基因的影响最小。结论腺相关病毒载体对关节软骨细胞中内参基因表达的影响低于质粒型载体,表明腺相关病毒对关节软骨细胞本身影响较小;GAPDH和TUBB基因可作为实验的内参基因校正目的基因的表达量。
Objective To investigate the effect of two transgenic vectors,plasmid and adeno-associated virus（AAV）,on transcription levels of mRNAs of housekeeping genes GAPDH,B2M,18S rRNA and TUBB in rabbit articular chondrocytes.Methods Rabbit articualr chondrocytes cultured in vitro were transfected with plasmid pcDNA6.2-EGFP and adeno-associated viral vector AAV2-EGFP,containing enhanced green fluorescent protein（EGFP） gene as reporter gene,respectively,and determined for expression of EGFP by fluorescent microscopy,and for transcription levels of GAPDH,B2M,18S rRNA and TUBB mRNAs by real-time PCR.Results Obvious green fluorescence was observed in articular chondrocytes 48 h after transfection with either pcDNA6.2-EGFP or AAV2-EGFP.The transcription levels of four housekeeping genes in two transgenic articular chondrocytes were lower than those in normal articular chondrocytes.However,the transcription levels of GAPDH and TUBB genes in the two transgenic articular chondrocytes were remarkably higher than those of B2M and 18S rRNA genes.AAV vector showed less effect on transgene.Conclusion Compared with plasmid,AAV vector showed less effect on the expressions of housekeeping genes in articular chondrocytes,indicating little effect of AAV on articular chondrocytes.GAPDH and TUBB genes might be used as internal reference gene for correction of expression level of target gene.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第6期697-699,703,共4页
Chinese Journal of Biologicals
基金
安徽省自然科学基金资助项目(11040606M163)
蚌埠医学院博士研究生科研启动基金(2010BR030)
关键词
转基因载体
软骨细胞
绿色荧光蛋白
内参基因
实时聚合酶链反应
Transgenic vector
Chondrocytes
Green fluorescent protein
Housekeeping gene
Real-time polymerase chain reaction
