腺病毒介导荧光素酶报告基因感染间充质干细胞的研究  

Infection with adenovirus-mediated luciferase reporter gene in mesenchymal stem cells and bioluminescence imaging

作　　者：王一帆[1][3] 夏睿[2] 郭玉林[3] 郜发宝[2] 

机构地区：[1]610041 成都,四川大学华西医院放射科分子影像研究室 [2]四川大学华西医院放射科分子影像研究室, 成都,610041 [3]宁夏医科大学总医院放射科

出　　处：《中华核医学与分子影像杂志》2013年第6期-,共5页Chinese Journal of Nuclear Medicine and Molecular Imaging

基　　金：国家重点基础研究计划(“973”计划)项目，国家自然科学基金

摘　　要：目的 构建携带萤火虫荧光素酶(Luc)报告基因的腺病毒载体(Ad-Luc),研究其感染大鼠骨髓间充质干细胞(BMSC)后的体内外生物发光成像.方法 从psiCHECK-2质粒中用PCR扩增Luc基因,克隆入腺病毒穿梭载体pShuttle-CMV后行Nhe Ⅰ/Xba Ⅰ双酶切和测序鉴定.重组腺病毒穿梭载体与骨架载体pAdeno同源重组并包装纯化后,测定其病毒滴度.用重组Ad-Luc感染BMSC,行体外生物发光成像确定最佳感染复数(MOI),并采用曲线拟合回归分析生物发光强度与MOI的关系.以锥虫蓝染色法评价细胞活力变化,计算细胞存活率.将转染后BMSC(1×106个)植入SD大鼠前肢肌肉内,行体内生物发光成像.细胞存活率组间比较采用两因素重复测量资料方差分析.结果 经酶切和测序鉴定证明,Ad-Luc构建成功,病毒滴度为1×1010空斑形成单位(PFU)/ml.体外生物发光检测结果显示最佳MOI值为50,Ad-Luc可高效感染BMSC,使其表达Luc,且拟合曲线示细胞生物发光强度随MOI增加而增强(R2 =0.98).转染组和未转染组细胞培养1、3、5、7d时,细胞存活率分别为(92.5±2.3)％与(94.1±1.8)％、(91.4±0.9)％与(92.7±2.0)％、(92.1±1.6)％与(93.3±2.4)％、(91.9±1.5)％与(93.0±3.1)％,2组间细胞活力的差异无统计学意义(F=4.38,P＞0.05).体内生物发光成像结果示BMSC移植1、3、7d后仍有存活,但随时间延长,生物发光信号逐渐减弱.结论 Luc报告基因通过腺病毒载体成功转入BMSC,实现了光学报告基因成像对移植干细胞的示踪.Objective To construct adenovirus vector containing firefly luciferase reporter gene (AdLuc) and infect bone marrow mesenchymal stem cells (BMSC),then to take bioluminescence imaging in vitro and in vivo for identification.Methods The luciferase gene was amplified with PCR from psiCHECK-2 plasmid and cloned into the adenoviral shuttle vector (pShuttle-CMV).It was confirmed by Nhe Ⅰ/Xba Ⅰ digestion and sequencing.PShuttle-CMV-Luc and backbone vector (pAdeno) were homologous recombined.Then the recombinant plasmid was packaged in HEK293 cells and the virus titer was detected.The BMSC were infected by the recombinant adenovirus.The bioluminescence imaging in vitro was performed to determine the best multiplicity of infection (MOI),and the relationship between bioluminescence intensity and MOI was analyzed by curve fitting regression analysis.Viability was evaluated via Trypan blue staining.The transfected BMSC (l× 106) were implanted into the muscles of forelimb of SD rats,and then tracked by bioluminescence imaging in vivo.Cell viability was compared using two-way repeated measures analysis of variance between groups.Results Enzyme digestion and sequence analysis indicated that Ad-Luc was successfully constructed.The virus titer was 1 × 1010 plaque forming unit (PFU)/ml.The bioluminescence detection in vitro showed that Ad-Luc could infect BMSC high efficiently to express luciferase and the best MOI was 50.The bioluminescence intensity enhanced with increase of MOI (R2 =0.98).No statistically significant difference was found in cell viability between transfected and untransfected BMSC at 1,3,5,7 d.The cell survival rates were (92.5±2.3)％ vs (94.1±1.8)％,(91.4±0.9)％ vs (92.7±2.0)％,(92.1±1.6)％ vs (93.3± 2.4) ％,(91.9 ± 1.5) ％ vs (93.0 ± 3.1) ％,respectively (F =4.38,P ＞ 0.05).The bioluminescence imaging in vivo showed that BMSC survived 1,3,7 d after implantation.However,bioluminescence signal decreased gradually over time.Conclusion It is feasible to apply the optical reporter gene imagi

关 键 词：骨髓 干细胞 腺病毒 人 荧光素酶 基因 报告 生物发光成像 大鼠 

分 类 号：R363[医药卫生—病理学]