2型重组腺相关病毒转染新西兰兔关节软骨细胞的体外研究

In vitro gene transfection into rabbit articular chondrocytes mediated by recombinant adeno-associated virus vector

目的为进一步研究腺相关病毒重组体在体内间接和直接基因治疗的效果提供依据。方法应用AAVMaxTM包装系统,复制和包装2型腺相关病毒重组体,进行纯化和滴度检测后,按照不同的转染指数(MOI)转染体外培养的新西兰兔关节软骨细胞,观察转染效果与及转染效果与时间的关系。倒置荧光显微镜观察证实表转染成功,流式细胞仪检测转染效果,包括表达绿色荧光信号细胞的百分比和平均荧光强度,将未转染的软骨细胞自发表达的绿色荧光信号百分比的最大值4·76%和荧光信号强度的最大值1·04设定为空白对照。结果当MOI值在1×105v·g·/cell以下时,MOI值(v·g·/cell)与转染率之间的剂量-反应曲线为正相关的线性关系,在MOI超过105v·g·/cell时,转染率不会再有显著的提高。在转染第7天,绿色荧光信号表达达到了峰值,随着转染时间的延长和传代次数的增加,表达绿色荧光信号的软骨细胞百分比和荧光强度均呈现降低趋势,但在转染后第56天仍可检测到绿色荧光蛋白的表达。结论2型腺相关病毒重组体在体外对新西兰兔关节软骨的转染效果良好;增强绿色荧光蛋白是观察2型腺相关病毒重组体转染关节软骨细胞效果一种良好的报告基因。

Objective To investigate the in vitro efficiency of recombinant adeno-associated virus （rAAV2） vector-mediated gene transfection into the rabbit articular chondrocytes. Methods Articular chondrocytes were isolated from New Zealand rabbits, cultured, and transfected with rAAV2 containing enhanced green fluorescent protein （eGFP） gene at different values multiplicity of infection （MOI）. Successful transfection of chondrocytes was confirmed by detection of recombinant enhanced green fluorescent protein using inverted fluorescence microscopy. The transfection efficiency was determined using the fluorescence-activated cell sorter. A maximum level of 4.76% was set as the background autofluorescence in live, uninfected chondrocytes. Results The percentage of eGFP-positive chondrocytes increased in a vector dose-dependent manner. When the MOI value was 〈 1 × 10^5 v. g./cell the transfection rate was positively linearly correlated with the dose. However, the MOI 〉 10^5 v. g. /cell did not markedly increase the transfection rate. The greatest population of eGFP-expressing cells was 97. 7% and the highest mean fluorescence intensity was 17.4 7 days after transfection. EGFP gene expression remained high until 56 days after transfection. Conclusion The in vitro efficiency of rAAV2 vector-mediated eGFP gene transfection into rabbit articular chondrocytes was high. The eGFP expression can be maintained for a rather long time. EGFP gene is an ideal tracer for chondrocytes transduction.