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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

Hits: 2188 Download times: 960 Received:December 26, 2018

作者	Author	单位	Unit	E-Mail
胡家铭	HU Jia-ming	浙江中医药大学附属第一医院浙江省中医院, 浙江杭州 310006 浙江中医药大学第一临床医学院, 浙江杭州 310006	The First Affiliated Hospital of Zhejiang Chinese Medical University, Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou 310006, Zhejiang, China
陈权	CHEN Quan	浙江中医药大学附属第一医院浙江省中医院, 浙江杭州 310006 浙江中医药大学第一临床医学院, 浙江杭州 310006	The First Affiliated Hospital of Zhejiang Chinese Medical University, Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou 310006, Zhejiang, China
周金依	ZHOU Jin-yi	浙江中医药大学附属第一医院浙江省中医院, 浙江杭州 310006 浙江中医药大学第一临床医学院, 浙江杭州 310006	The First Affiliated Hospital of Zhejiang Chinese Medical University, Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou 310006, Zhejiang, China
吴锦婷	WU Jin-ting	浙江中医药大学附属第一医院浙江省中医院, 浙江杭州 310006 浙江中医药大学第一临床医学院, 浙江杭州 310006	The First Affiliated Hospital of Zhejiang Chinese Medical University, Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou 310006, Zhejiang, China
余欢	YU Huan	浙江中医药大学附属第一医院浙江省中医院, 浙江杭州 310006 浙江中医药大学第一临床医学院, 浙江杭州 310006 浙江中医药大学附属第一医院骨伤研究所, 浙江杭州 310006	The First Affiliated Hospital of Zhejiang Chinese Medical University, Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou 310006, Zhejiang, China
付方达	FU Fang-da	浙江中医药大学附属第一医院浙江省中医院, 浙江杭州 310006 浙江中医药大学第一临床医学院, 浙江杭州 310006 浙江中医药大学附属第一医院骨伤研究所, 浙江杭州 310006	The First Affiliated Hospital of Zhejiang Chinese Medical University, Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou 310006, Zhejiang, China
阮红峰	RUAN Hong-feng	浙江中医药大学附属第一医院浙江省中医院, 浙江杭州 310006 浙江中医药大学附属第一医院骨伤研究所, 浙江杭州 310006 浙江中医药大学, 浙江杭州 310053 上海中医药大学附属龙华医院, 上海 200032	The First Affiliated Hospital of Zhejiang Chinese Medical University, Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou 310006, Zhejiang, China	rhf@zcmu.edu.cn
肖鲁伟	XIAO Lu-wei	浙江中医药大学附属第一医院浙江省中医院, 浙江杭州 310006 浙江中医药大学第一临床医学院, 浙江杭州 310006 浙江中医药大学附属第一医院骨伤研究所, 浙江杭州 310006 浙江中医药大学, 浙江杭州 310053	The First Affiliated Hospital of Zhejiang Chinese Medical University, Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou 310006, Zhejiang, China
吴承亮	WU Cheng-Liang	浙江中医药大学附属第一医院浙江省中医院, 浙江杭州 310006 浙江中医药大学第一临床医学院, 浙江杭州 310006 浙江中医药大学附属第一医院骨伤研究所, 浙江杭州 310006 浙江中医药大学, 浙江杭州 310053	The First Affiliated Hospital of Zhejiang Chinese Medical University, Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou 310006, Zhejiang, China
童培建	TONG Pei-jian	浙江中医药大学附属第一医院浙江省中医院, 浙江杭州 310006 浙江中医药大学第一临床医学院, 浙江杭州 310006 浙江中医药大学附属第一医院骨伤研究所, 浙江杭州 310006	The First Affiliated Hospital of Zhejiang Chinese Medical University, Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou 310006, Zhejiang, China

期刊信息：《中国骨伤》2019年32卷，第8期，第750-755页

DOI：10.3969/j.issn.1003-0034.2019.08.015

基金项目:国家自然科学基金项目（编号：81804121，81573994，81273770）；国家博士后基金面上项目（编号：2018M632154）；浙江省自然科学基金（编号：LY19H270006，LQ17H270005，LY15H270012）；浙江省中医药卫生科技计划项目（编号：2017ZB026）；浙江省大学生科技创新活动计划（新苗人才计划）项目（编号：2018R410030）；浙江省医学会临床科研基金项目（编号：2017ZYC-A23）

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（6 μl）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1×10¹³ vg/ml），维5组（维真生物AAV5，滴度为1×10¹³ vg/ml），维6组（维真生物AAV6，滴度为1×10¹³ vg/ml），维7组（维真生物AAV7，滴度为1×10¹³ vg/ml），维8组（维真生物AAV8，滴度为1×10¹³ vg/ml），维9组（维真生物AAV9，滴度为1×10¹³ vg/ml），汉DJ组（汉恒生物AAV2-DJ，滴度为1×10¹² vg/ml），汉5组（汉恒生物AAV5，滴度为1×10¹² 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10 μm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。

[关键词]:腺病毒血清膝关节软骨细胞感染

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

Abstract:

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(6 μl) 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 1×10¹³ vg/ml),Vigene 5 group (AAV5 from vigene biosciences,titer for 1×10¹³ vg/ml),Vigene 6 group (AAV6 from vigene biosciences,titer for 1×10¹³ vg/ml),Vigene 7 group (AAV7 from vigene biosciences,titer for 1×10¹³ vg/ml),Vigene 8 group (AAV8 from vigene biosciences,titer for 1×10¹³ vg/ml),Vigene 9 group (AAV9 from vigene biosciences,titer for 1×10¹³ vg/ml),Hanbio DJ group(AAV2-DJ from Hanbio,titer for 1×10¹² vg/ml),Hanbio 5 group (AAV5 from Hanbio,titer for 1×10¹² 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 10 μm 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.

KEYWORDS:Adenoviruses Serum Knee joint Chondrocytes Injection

引用本文，请按以下格式著录参考文献：

中文格式:	胡家铭,陈权,周金依,吴锦婷,余欢,付方达,阮红峰,肖鲁伟,吴承亮,童培建.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[J].中国骨伤,2019,32(8):750~755
英文格式:	HU Jia-ming,CHEN Quan,ZHOU Jin-yi,WU Jin-ting,YU Huan,FU Fang-da,RUAN Hong-feng,XIAO Lu-wei,WU Cheng-Liang,TONG Pei-jian.Comparative study on infection rate of different adeno-associated virus for knee joint cartilage in mice[J].zhongguo gu shang / China J Orthop Trauma ,2019,32(8):750~755

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