增强绿色荧光蛋白标记比较腺病毒、腺相关病毒体外转染兔软骨细胞的效果

The comparation of transfection efficiency to New Zealand rabbit chondrocytes using enhanced green fluorescent protein in vitro

目的通过增强绿色荧光蛋白（eGFP）标记，比较腺病毒（Ad）、腺相关病毒（AAV）对体外培养软骨细胞的转染效果。方法体外培养正常3月龄新西兰兔关节软骨细胞，以rAd5-eGFP、rAAV2-eGFP分别转染原代关节软骨细胞，计算最佳传染复数。之后以最佳传染复数的病毒重组体转染软骨细胞，分别应用流式细胞仪检测绘制转染率的时间-反应曲线，倒置荧光显微镜下观察转染后细胞的大体形态、绿色荧光的表达情况并应用荧光定量PCR检测转染前后软骨细胞Ⅱ型胶原mRNA表达水平的变化。结果rAd5-eGFP、rAAV2-eGFP的最佳传染复数分别为1×10^3vp／cell和1×10^5vg／cell。分别应用最佳传染复数体外转染原代培养的软骨细胞后，rAd5-eGFP在转染后1～2d荧光表达即可达到高峰，但之后迅速衰减，在转染后第28天基本检测不到荧光表达。rAAV2-eGFP在转染第7天荧光表达达高峰，随之缓慢衰减，在转染后第56天仍可检测到绿色荧光表达。荧光定量PCR检测显示，rAd5-eGFP在转染软骨细胞后，软骨细胞合成Ⅱ型胶原mRNA的水平明显下降，而rAAV2-eGFP则影响不显著。结论Ad转染体外培养的关节软骨细胞后，目的基因的表达迅速，但维持时间短，对软骨细胞表型的影响较明显；AAV转染软骨细胞后，目的基因的表达缓慢，但维持时间长，且对软骨细胞表型的影响较小。AAV更适于作为软骨细胞的体外转染载体。

Objective To compare the transfection efficiency of recombinant adeno-associated virus （rAAV） vector and adenovirus（rAd） vector to rabbit articular chondrocytes in vitro. Methods Primarily isolated rabbit articular chondrocytes were infected at various multiplicities of infection （MOI） respectively with rAAV2 and rAd5 containing the enhanced green fluorescent protein（eGFP） gene. The transfection efficiency was determined with use of the fluorescence-activated cell sorter. Inverted fluorescence microscopy was used to dected gene expression of eGFP and chondrocytes shape. MRNA expressions of type Ⅱ collagen were determined by quantitative real-time PCR to assess the toxicity of rAAV2 and tAd5. Results The best trans- fection efficiency of rAAV2 and tAd5 was 1×10^5 vg/cell and 1×10^3 vp/cell respectively. Enhanced green fluorescent reached to the peak ＇after one or two days, but then decayed rapidly and was barely detected 28 d late when primarily isolated rabbit articular chondrocytes were infected at the best MOI with fAdS. As to rAAV2, enhanced green fluorescent reach to the peak 7 d late, decayed slowly and was detected 56 d late when primarily isolated rabbit articular chondrocytes were infected at the best MOI. Quantitative real-time PCR showed that decreases in collagen Ⅱ mRNA were significantly greater in the chondrocytes infected with tad5 than rAAV2. Conclusion Compared with tad5, rAAV2 vector is ideal in transfection efficiency maintaining and safty to New Zealand rabbit chondrocytes in vitro.