腺病毒介导的生长分化因子5基因对hBMSCs成骨分化的影响  

EFFECT OF GROWTH AND DIFFERENTIATION FACTOR 5 GENE TRANSFERRED BY ADENOVIRUS ON hBMSCs OSTEOGENIC DIFFERENTIATION

作　　者：程相俊[1] 刘浩[1] 张霆[2] 黄巧蓉[2] 

机构地区：[1]四川大学华西医院骨科,成都610041 [2]四川大学华西医院生物治疗国家重点实验室,成都610041

出　　处：《中国修复重建外科杂志》2009年第8期985-990,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30572163)~~

摘　　要：目的利用重组腺病毒载体将生长分化因子5(growth and differentiation factor5,GDF-5)基因导入hBMSCs,研究GDF-5基因的表达和对hBMSCs成骨分化的影响。方法将重组腺病毒GDF-5(adenovirus GDF-5,Ad-GDF-5)[含绿色荧光蛋白(green fluorescent protein,GFP)标记]和空载腺病毒Ad-GFP进行扩增并测定滴度,并以不同滴度两种病毒感染第3代hBMSCs,干预后2d荧光显微镜下观察GFP表达情况,RT-PCR检测GDF-5在hBMSCs中的表达。取第3代贴壁hBMSCs随机分为4组:实验组(GDF-5基因转染组)、成骨诱导组、空载组和对照组。于干预后不同时间行倒置相差显微镜观察、荧光显微镜观察、荧光定量RT-PCR、免疫荧光染色和vonKossa染色检测细胞分化情况。结果Ad-GDF-5滴度为1.0×109pfu/mL,Ad-GFP滴度为1.2×109pfu/mL。Ad-GDF-5及Ad-GFP均能对hBMSCs有效感染,并使其表达目的基因和GFP基因。干预后1～7d实验组和成骨诱导组hBMSCs细胞形态及生长方式向成骨细胞转化,而对照组和空载组hBMSCs仍保持原有形态及生长方式。荧光定量RT-PCR检测示,干预后4d实验组GDF-5基因表达明显高于其余各组(P<0.05);实验组和成骨诱导组ALP、ColⅠ、骨钙素基因表达均高于空载组和对照组(P<0.05),成骨诱导组ColⅠ基因表达高于实验组(P<0.05)。干预后4d,免疫荧光染色示成骨诱导组和实验组细胞表达并分泌ColⅠ,空载组和对照组细胞未见ColⅠ表达。干预后10d,成骨诱导组和实验组细胞vonKossa染色为阳性,对照组和空载组为阴性。结论GDF-5基因可通过腺病毒载体导入hBMSCs稳定表达,并促使其成骨分化,为进一步研究这种转基因hBMSCs修复骨组织奠定了基础。Objective To introduce growth and differentiation factor 5 （GDF-5） gene into hBMSCs using recombinant adenovirus vector and to investigate the effect of GDF-5 gene expression on hBMSCs osteogenic differentiation. Methods Recombinant adenovirus GDF-5 （Ad-GDF-5） containing green fluorescent protein （GFP） and Ad-GFP were amplified and tittered, hBMSCs at passage 3 were infected with two viruses at different titers. At 2 days after intervention, GFP expression was observed using fluorescence microscope, and GDF-5 expression in hBMSCs was detected by RT-PCR. Adherent hBMSCs at passage 3 were randomly divided into 4 groups： experimental group （GDF-5 gene transfection）, osteogenic induction group, Ad- GFP infection group, and control group. Cell differentiation was detected by inverted phase contrast microscope observation, fluorescence microscope observation, reverse transcription fluorescence quantitative PCR, immunofluorescence staining, and von Kossa staining at different time points after intervention. Results The titer of Ad-GDF-5 and Ad-GFP was 1.0 × 10^9 pfu/mL and 1.2 × 10^9 pfu/mL, respectively, hBMSCs was efficiently infected by Ad-GDF-5 and Ad-GFP, and expressed target gene and GFP gene. At 1-7 days after intervention, morphology and growth pattern of the hBMSCs in the experimental group and the osteogenic induction group were transformed into osteoblast-tike cells, whereas the cells in the other two groups were still maintained their original morphology and growth pattern. Reverse transcription fluorescence quantitative PCR detection： at 4 days after intervention, GDF-5 expression in the experimental group was obviously higher than that of other groups （P 〈 0.05）; ALP, Col Ⅰ, and OC gene expression in the experimental and the osteogenic induction group were superior to those of the Ad-GFP infection and the control group （P 〈 0.05）; Col Ⅰ gene expression in the osteogenic induction group was greater than that of the experimental group （P 〈 0.05）. Immunofluorescence

关 键 词：生长分化因子5 HBMSCS 重组腺病毒 分化 基因治疗 

分 类 号：R68[医药卫生—骨科学]