中华神经医学杂志
中華神經醫學雜誌
중화신경의학잡지
CHINESE JOURNAL OF NEUROMEDICINE
2013年
5期
433-438
,共6页
郭永坤%张业森%刘春颖%戴宜武%黄瑾%姚慧%吴炳山%姚学勤%杨志军
郭永坤%張業森%劉春穎%戴宜武%黃瑾%姚慧%吳炳山%姚學勤%楊誌軍
곽영곤%장업삼%류춘영%대의무%황근%요혜%오병산%요학근%양지군
转铁蛋白受体%慢病毒载体%神经干细胞%增强型绿色荧光蛋白
轉鐵蛋白受體%慢病毒載體%神經榦細胞%增彊型綠色熒光蛋白
전철단백수체%만병독재체%신경간세포%증강형록색형광단백
Human transferrin receptor%Lentiviral vector%Neural stem cell%Enhanced green fluorescence protein
目的 探讨人转铁蛋白受体(hTfR)基因慢病毒载体构建方法及其在神经干细胞(NSCs)中的表达情况,为NSCs的MR分子成像提供实验基础. 方法 利用聚合酶链反应技术(PCR)扩增hTfR基因,并克隆到pLenti6.3载体,构建出慢病毒表达载体pLentiI6.3-hTfR-IRES-EGFP.利用Lipofectin2000试剂将PLP1、PLP2、PLP-VSVG和pLenti6.3-hTfR-IRES-EGFP共转染293T细胞进行慢病毒包装,48 h后收集病毒上清,体外感染NSCs.细胞流式筛选稳定表达hTfR的细胞,通过实时定量PCR和Western boltting检测hTfR的表达,细胞免疫荧光技术对过表达的hTfR进行亚细胞的定位. 结果 成功构建hTfR基因慢病毒表达载体,包装的慢病毒颗粒成功感染NSCs.实时定量PCR和Western boltting鉴定出hTfR在NSCs中过表达,hTfR基因表达相对值为2.275±0.281.细胞免疫荧光检测到过表达的hTfR主要在细胞膜上表达.NSCs分化后,hTfR在胶质细胞和神经元中稳定表达. 结论 本研究所用方法能成功构建hTfR慢病毒表达载体并筛选出稳定表达hTfR的NSCs系,为下一步活体内移植NSCs行MR分子成像实验研究奠定了基础.
目的 探討人轉鐵蛋白受體(hTfR)基因慢病毒載體構建方法及其在神經榦細胞(NSCs)中的錶達情況,為NSCs的MR分子成像提供實驗基礎. 方法 利用聚閤酶鏈反應技術(PCR)擴增hTfR基因,併剋隆到pLenti6.3載體,構建齣慢病毒錶達載體pLentiI6.3-hTfR-IRES-EGFP.利用Lipofectin2000試劑將PLP1、PLP2、PLP-VSVG和pLenti6.3-hTfR-IRES-EGFP共轉染293T細胞進行慢病毒包裝,48 h後收集病毒上清,體外感染NSCs.細胞流式篩選穩定錶達hTfR的細胞,通過實時定量PCR和Western boltting檢測hTfR的錶達,細胞免疫熒光技術對過錶達的hTfR進行亞細胞的定位. 結果 成功構建hTfR基因慢病毒錶達載體,包裝的慢病毒顆粒成功感染NSCs.實時定量PCR和Western boltting鑒定齣hTfR在NSCs中過錶達,hTfR基因錶達相對值為2.275±0.281.細胞免疫熒光檢測到過錶達的hTfR主要在細胞膜上錶達.NSCs分化後,hTfR在膠質細胞和神經元中穩定錶達. 結論 本研究所用方法能成功構建hTfR慢病毒錶達載體併篩選齣穩定錶達hTfR的NSCs繫,為下一步活體內移植NSCs行MR分子成像實驗研究奠定瞭基礎.
목적 탐토인전철단백수체(hTfR)기인만병독재체구건방법급기재신경간세포(NSCs)중적표체정황,위NSCs적MR분자성상제공실험기출. 방법 이용취합매련반응기술(PCR)확증hTfR기인,병극륭도pLenti6.3재체,구건출만병독표체재체pLentiI6.3-hTfR-IRES-EGFP.이용Lipofectin2000시제장PLP1、PLP2、PLP-VSVG화pLenti6.3-hTfR-IRES-EGFP공전염293T세포진행만병독포장,48 h후수집병독상청,체외감염NSCs.세포류식사선은정표체hTfR적세포,통과실시정량PCR화Western boltting검측hTfR적표체,세포면역형광기술대과표체적hTfR진행아세포적정위. 결과 성공구건hTfR기인만병독표체재체,포장적만병독과립성공감염NSCs.실시정량PCR화Western boltting감정출hTfR재NSCs중과표체,hTfR기인표체상대치위2.275±0.281.세포면역형광검측도과표체적hTfR주요재세포막상표체.NSCs분화후,hTfR재효질세포화신경원중은정표체. 결론 본연구소용방법능성공구건hTfR만병독표체재체병사선출은정표체hTfR적NSCs계,위하일보활체내이식NSCs행MR분자성상실험연구전정료기출.
Objective To clone the human transferrin receptor (hTfR) gene into the lentiviral expression vector plenti6.3,identify the reconstructed plasmid and detect the expression ofhTfR gene in neural stem cells (NSCs),which will provide experimental foundation for in vivo NSCs MR molecular imaging.Methods The hTfR gene cDNA was amplified by polymerase chain reaction (PCR) and cloned into the pLenti6.3 vector to generate the lentiviral vector pLenti6.3-hTfR-IRES-EGFP.The lentiviral vector system (PLP1,PLP2,PLP-VSVG and pLentiI6.3-hTfR-IRES-EGFP) was co-transfected into 293T cells with lipofectin 2000 reagent.The packaged viruses were harvested 48 h later.NSCs were infected by lentivirus carrying hTfR reporter gene.Transfected cells were screened by flow cytometry (FCM).The expressions ofhTfR was confirmed by real-time quantitative PCR and Western blotting.The location ofhTfR gene was detected by cell immunofluorescence.Results Lentivirus vector containing hTfR gene was successfully constructed.NSCs could be infected by the recombinant lentivirus.The over-expressions of hTfR could be successfully detected in the infected NSCs by real-time quantitative PCR (the relative value was 2.275 ±0.281) and Western blotting.Cell immunofluorescence displayed that hTfR was predominantly located on cytomembrane; the expressions ofhTfR were not affected after NSCs differentiating into glial cells and neuuronal cells.Conclusion Lentiviral vector pLenti6.3-hTfR-IRES-EGFP is successfully constructed and infected to the primary cultured NSCs,and a NSC cell line stablely expressed hTfR is successfully screened,which paves the way for further research on MR molecular imaging of NSCs transplantation in vivo.
