不同血清型腺相关病毒对小鼠膝关节软骨细胞感染效率的比较研究

Comparative study on infection rate of different adeno-associated virus for knee joint cartilage in mice

目的:比较不同血清型腺相关病毒(adeno-associated virus,AAV)对小鼠膝关节软骨细胞的感染效率,寻找理想的膝关节软骨细胞基因编辑工具。方法:选取4周龄C57BL/6雄鼠45只,体重(14.3±0.2)g,根据右侧膝关节腔注射试剂(6μl)的不同分为以下9组,每组5只。对照组(生理盐水),维2组(维真生物AAV2,滴度为1×10^13 vg/ml),维5组(维真生物AAV5,滴度为1×10^13 vg/ml),维6组(维真生物AAV6,滴度为1×10^13 vg/ml),维7组(维真生物AAV7,滴度为1×10^13 vg/ml),维8组(维真生物AAV8,滴度为1×10^13 vg/ml),维9组(维真生物AAV9,滴度为1×10^13 vg/ml),汉DJ组(汉恒生物AAV2-DJ,滴度为1×10^12 vg/ml),汉5组(汉恒生物AAV5,滴度为1×10^12 vg/ml)。所有病毒过表达绿色荧光蛋白(GFP)。在注射30 d后取膝关节,于体视显微镜下大体观察软骨损伤情况后,做10μm冰冻切片,采用荧光显微镜观察膝关节软骨和半月板绿色荧光分布情况。结果:体视显微镜观察结果表明,本研究采取的膝关节腔注射方法对小鼠软骨无明显损伤。组织切片的荧光观察显示,经不同血清型AAV注射后的小鼠膝关节软骨细胞均发出较强的绿色荧光,荧光强度分析中以维2组和维7组软骨和半月板的荧光强度最高,平均光密度值分别为0.077±0.020和0.061±0.022,上述2组与其他各组间比较差异有统计学意义。结论:通过在体膝关节腔注射AAV可成功感染小鼠膝关节软骨细胞,AAV2和AAV7对膝关节软骨组织感染效率高且特异性较强,该方法可作为膝关节软骨细胞基因编辑的有效工具。

Objective:To explore infection rate of different adeno-associated virus(AAV)on knee joint cartilage in mice and to find a good gene editing tool for mice chondrocytes of knee joint.Methods:Forty-five 4-week-old SPF C57BL/6 weighed(14.3±0.2)g were selected.According to different injections(6μl)for right knee joint,mice were divided into 9 different groups,5 mice in each group.The groups were such as following:control group(normal saline),Vigene 2 group(AAV2 from vigene biosciences,titer for 1×10^13 vg/ml),Vigene 5 group(AAV5 from vigene biosciences,titer for 1×10^13 vg/ml),Vigene 6 group(AAV6 from vigene biosciences,titer for 1×10^13 vg/ml),Vigene 7 group(AAV7 from vigene biosciences,titer for 1×10^13 vg/ml),Vigene 8 group(AAV8 from vigene biosciences,titer for 1×10^13 vg/ml),Vigene 9 group(AAV9 from vigene biosciences,titer for 1×10^13 vg/ml),Hanbio DJ group(AAV2-DJ from Hanbio,titer for 1×10^12 vg/ml),Hanbio 5 group(AAV5 from Hanbio,titer for 1×10^12 vg/ml).All AAVs were over-expressed green fluorescent protein(GFP).Knee joint specimens were taken and observed injury of cartilage under stereomicroscope at 30 days after injection,then 10μm thick frozen sections were prepared.Distribution of green fluorescent protein of meniscus and cartilage of knee joint was observed under fluorescence microscope.Results:Stereomicroscope observation indicated that no obvious lesion was observed in knee joint cartilage of mice after intra-articular injection of AAV.According to frozen sections of knee joints,strong green fluorescence was observed in knee joint cartilage in all AAV experimental groups.Compared with other groups,significantly stronger green fluorescence were observed both in AAV2 and AAV7 groups,whose average fluorescence density was 0.077±0.020 and 0.061±0.022.There were significant differences between two groups and other groups.Conclusion:AAV could infect chondrocyte of knee joint in vivo by injecting into knee joint cavity.Higher infection efficiency of AAV2 and AAV7 on knee joint cartilage were observed.Local injection of AAV into knee joint cavity could be used as an effective tool for gene editing of knee joint chondrocyte.