腺病毒载体携带HSV1-TK报告基因感染BMSCs后摄取^(131)I-FIAU的实验研究  

Uptake of ^(131)I-FIAU in BMSCs infected by adenovirus vector-mediated HSV1-TK

作　　者：张斌青[1] 吴涛[1] 孙逊[1] 安锐[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院核医学科湖北省分子影像重点实验室,武汉430022

出　　处：《核技术》2010年第4期303-307,共5页Nuclear Techniques

基　　金：国家自然科学基金资助(30770605)资助

摘　　要：用内部核糖体进入序列(IRES)将报告基因单纯疱疹I型病毒胸苷激酶(HSV1-TK)和治疗基因脑源性神经营养因子(BDNF)连接,双启动子法将TK-IRES-BDNF与增强绿色荧光蛋白(EGFP)包装到重组腺病毒载体中,得到Ad5-TK-IRES-BDNF-EGFP,扩增、纯化、测定病毒滴度;以感染复数(MOI)为0、50、100、150、200、250感染体外培养的骨髓间充质干细胞(BMSCs),以重组腺病毒Ad5-EGFP为无效对照病毒。荧光显微镜下观察感染细胞的绿色荧光细胞阳性率。四甲基偶氮唑蓝(MTT)比色法检测感染细胞增殖能力。碱性成纤维细胞生长因子(bFGF)和表皮生长因子(EGF)诱导感染细胞向神经元细胞分化,显微镜下观察细胞形态变化。实时定量聚合酶链反应(RQ-PCR)和2-△△CT法分析两目的基因的相对表达量(CT)。观察感染细胞对131I-FIAU的摄取情况。重组腺病毒Ad5-TK-IRES-BDNF-EGFP在MOI为150可高效感染BMSCs,感染细胞存活率>98%,且保持良好的神经元细胞诱导分化能力。RQ-PCR检测两目的基因随着腺病毒MOI增加表达增强,且TK和BNDF两基因的表达有良好的线性相关(r=0.973,P<0.05,n=3)。在前3h可见感染目的基因组细胞对131I-FIAU的摄取程度与时间呈正相关,3h感染目的基因组对131I-FIAU摄取率可达(31.42±0.46)%(n=3),随后增加不明显。各点感染目的基因组摄取率均显著高于对照组(t=23.06–173.83,P<0.05,n=3)。重组腺病毒载体能高效、低毒感染BMSCs,IRES介导两目的基因表达有良好的线性相关。本研究表明感染目的基因细胞可有效介导HSV1-TK摄取131I-FIAU,为后续放射性核素报告基因活体显像示踪转基因BMSCs治疗脑梗死提供了细胞水平依据。Report gene HSV1-TK and therapy gene were connected by IRES, and recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP was constructed and infected with BMSCs at MOI of 0, 50, 100, 150, 200 and 250, with the control recombinant adenovirus vector of Ad5-EGFE Green fluorescence cell positive rate was observed under the microscopy. MTT assay was used to determine the cell proliferation, bFGF and EGF were used to induce the BMSCs, and RQ-PCR to determine target gene expression in infection BMSCs. Uptake of ^131I-FIAU was assessed by gamma counter. The data were processed by SPSS11.code. Recombinant adenovirus at MOI 150 had high infection efficiency and low toxic in BMSCs. There was a strong relation between the mRNA expression of TK and BDNF in infection BMSCs. The significance between the infection BMSCs and control BMSCs for uptake of ^131I-FIAU at all the time points was t=-23.06-173.83 and P〈0.05 （n=3）. Recombinant adenovirus vector had high infection and low toxic for BMSCs, and effective uptake of ^131I-FIAU. This suggests a suitable gene vector for tracing genetically modified stem cells.

关 键 词：报告基因 干细胞 基因治疗 腺病毒 

分 类 号：R817.4[医药卫生—影像医学与核医学]