利用报告基因rNIS监测大鼠骨髓间充质干细胞移植大鼠心肌的可行性研究  

Study of rNIS as a reporter gene monitoring rBMSC transplanted to rat myocardium

作　　者：胡硕[1] 兰晓莉[1] 曹卫[1] 曹国祥[1] 张国鹏[1] 张斌青[1] 吴涛[1] 常伟[1] 张永学[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：《中华核医学杂志》2010年第3期180-184,共5页Chinese Journal of Nuclear Medicine

基　　金：基金项目：国家自然科学基金（30772208,30830041）;中国博士后科学基金（20090451053）;湖北省分子影像重点实验室开放基金（2009027）

摘　　要：目的 研究大鼠钠碘转运体（rNIS）作为报告基因监测大鼠骨髓间充质干细胞（rBMSC）移植至大鼠心肌的可行性.方法 构建含rNIS/增强绿色荧光蛋白（EGFP）基因的重组腺病毒载体（Ad/rNIS/EGFP）,转染rBMSC后应用荧光显微镜观察细胞表达EGFP情况.按随机数字表法将健康大鼠分为3组,rNIS组大鼠心肌内移植转染rNIS的rBMSC 抑制组大鼠心肌内移植转染rNIS的rBMSC,γ显像前口服高氯酸纳 对照组大鼠心肌内移植未转染病毒的rBMSC.细胞移植到大鼠心肌后,以99TcmO4-作为报告探针对大鼠进行γ显像,随后进行生物学分布实验,用实时聚合酶链反应（PCR）和Western blot分别检测心肌组织中rNIS基因和蛋白表达水平,免疫组织化学检测心肌组织中CD11b、CD29、CD34、CD44、CD45和CD90表达情况.组间计量资料的差异用t检验分析.结果 重组腺病毒转染后,荧光显微镜下可见到高表达EGFP的rBMSC.rNIS转染的rBMSC移植到大鼠心肌后,γ显像能清晰显示移植部位的心肌,心肌/右前肢放射性比值（RUR）为6.7±0.4,对照组RUR为3.0±0.2,二者之间比较差异有统计学意义,t=2.78,P=0.03.生物学分布实验中,对照组心肌组织的每克组织百分注射剂量率（%ID/g）为2.5±0.4,明显低于rNIS组的60.2±20.8,差异有统计学意义,t=7.13,P＜0.001.移植心肌组织中rNIS的基因和蛋白都高表达,而对照组心肌组织rNIS表达较低.移植转染细胞的心肌组织中,CD29、CD44、CD90表达阳性,CD11b表达弱阳性,CD34、CD45表达阴性.结论 rNIS可作为报告基因有效监测rBMSC移植大鼠心肌.Objective To investigate the feasibility of rat sodium/iodide symporter （rNIS） as a reporter gene monitoring rat bone marrow mesenchymal cells （rBMSC） transplanted to rat myocardium in vivo.Methods Recombinated adenovirus vector was constructed by rNIS/enhanced green fluorescence protein （EGFP） （Ad-rNIS/EGFP）.rBMSC transfected by Ad-rNIS/EGFP were studied using fluorescence microscope.Fifteen rats were transplanted with rBMSC and randomly divided into three groups：rNIS group （with rNIS transfection）, blocked group （with rNIS transfection） by oral intake of perchloric sodium before planar imaging（GE Millennium MPR SPECT）, and control group （without rNIS transfection）.All rats underwent 99Tcm-pertechnetate planar imaging.The biological distribution of 99Tcm-pertechnetate was studied.The expressions of rNIS gene and protein in myocardium were measured by real time polymerase chain reaction （PCR） and western blot, respectively.The expressions of CD29, CD44, CD90, CD11b, CD34 and CD45 were measured by immunohistochemistry.Results rBMSC transfected by Ad-rNIS/EGFP showed EGFP expression under fluorescence microscope.The transplanted rat myocardium could be visualized on 99Tcm-pertechnetate planar imaging in rNIS group.The relative uptake ratio（ Rheart/Rhmb, RUR） was 6.7 ±0.4.RUR in control group （3.0 ±0.2） was lower than that in rNIS group （t =2.78, P=0.03）.The percentage injection dose per gram of tissue （% ID/g） of the transplanted myocardium was 60.2 ± 20.8 in rNIS group,which was higher than that （2.5 ± 0.4） % ID/g of control group （ t = 7.13, P〈0.001 ）.rNIS gene and protein were highly expressed in transplanted myocardium in rNIS group but less expressed in control group.The expressions of CD29, CD44 and CD90 were positive, CD45 and CD45 negative CD11b mildly positive in the myocardium transplanted with infective rBMSC.Conclusion rNIS can efficiently monitor rBMSC transplanted to rat myocardium.

关 键 词：大鼠 心肌 骨髓 干细胞 移植 钠碘转运体 放射性核素显像 

分 类 号：R541[医药卫生—心血管疾病]