CAJ | 학술논문

目的通过制备携带NIS及EGFP基因的重组慢病毒并转染骨髓间充质干细胞(BMSCs),探讨NIS基因监测BMSCs向颅内脑胶质瘤迁移的可行性.方法将NIS及EGFP基因片段分别亚克隆至慢病毒载体pLVX-puro,制备慢病毒pLVX-CMV-NIS-EGFP,同时构建对照组慢病毒pLVX-CMV-0-EGFP.分离培养BMSCs,转染后经嘌呤霉素筛选,得到稳定干细胞株BMSCs-NIS-EGFP及对照组干细胞株BMSCs-EGFP.Western blot分析NIS蛋白在细胞中的表达.通过摄碘实验、NaClO4抑制实验验证NIS蛋白的功能.制备裸鼠颅内脑胶质瘤模型,经尾静脉注入BMSCs-NIS-EGFP,24h后行125I microSPECT显像,观察NIS作为报告基因监测BMSCs-NIS-EGFP向颅内脑胶质瘤迁移的情况.结果成功构建并制备重组慢病毒pLVX-CMV-NIS-EGFP及pLVX-CMV-0-EGFP,完成BMSCs的分离培养.病毒转染经嘌呤霉素筛选得到稳定于细胞株BMSCs-NIS-EGFP及BMSCs-EGFP.Western blot显示NIS基因在BMSCs-NIS-EGFP中大量表达,而在BMSCs-EGFP中无表达.细胞摄碘实验显示BMSCs-NIS-EGFP细胞具有摄碘功能,30 min摄碘峰值是对照组BMSCs-EGFP细胞的近10倍,且其摄碘可被NaClO4显著抑制.成功制备裸鼠脑胶质瘤模型,microSPECT显像显示颅内脑胶质瘤部位有明显的125I放射性摄取增高.结论重组慢病毒pLVX-CMV-NIS-EGF可使NIS基因在BMSCs中表达并介导125I的摄取,NIS基因可有效监测BMSCs向颅内脑胶质瘤的迁移,这为开展以BMSCs为载体、由NIS介导的脑胶质瘤基因靶向治疗奠定了基础.
목적 통과제비휴대NIS급EGFP기인적중조만병독병전염골수간충질간세포(BMSCs),탐토NIS기인감측BMSCs향로내뇌효질류천이적가행성.방법 장NIS급EGFP기인편단분별아극륭지만병독재체pLVX-puro,제비만병독pLVX-CMV-NIS-EGFP,동시구건대조조만병독pLVX-CMV-0-EGFP.분리배양BMSCs,전염후경표령매소사선,득도은정간세포주BMSCs-NIS-EGFP급대조조간세포주BMSCs-EGFP.Western blot분석NIS단백재세포중적표체.통과섭전실험、NaClO4억제실험험증NIS단백적공능.제비라서로내뇌효질류모형,경미정맥주입BMSCs-NIS-EGFP,24h후행125I microSPECT현상,관찰NIS작위보고기인감측BMSCs-NIS-EGFP향로내뇌효질류천이적정황.결과 성공구건병제비중조만병독pLVX-CMV-NIS-EGFP급pLVX-CMV-0-EGFP,완성BMSCs적분리배양.병독전염경표령매소사선득도은정우세포주BMSCs-NIS-EGFP급BMSCs-EGFP.Western blot현시NIS기인재BMSCs-NIS-EGFP중대량표체,이재BMSCs-EGFP중무표체.세포섭전실험현시BMSCs-NIS-EGFP세포구유섭전공능,30 min섭전봉치시대조조BMSCs-EGFP세포적근10배,차기섭전가피NaClO4현저억제.성공제비라서뇌효질류모형,microSPECT현상현시로내뇌효질류부위유명현적125I방사성섭취증고.결론 중조만병독pLVX-CMV-NIS-EGF가사NIS기인재BMSCs중표체병개도125I적섭취,NIS기인가유효감측BMSCs향로내뇌효질류적천이,저위개전이BMSCs위재체、유NIS개도적뇌효질류기인파향치료전정료기출.
Objective To construct a recombinant lentiviral expression vector containing NIS and EGFP gene,and to explore the feasibility of NIS gene for monitoring the bone marrow derived mesenchymal stem cells (BMSCs) migration to the intracranial glioma.Methods The NIS and EGFP gene fragments were subcloned into lentiviral vector pLVX-puro,then packaged and amplified in HEK293T cells to obtain recombinant lentivirus pLVX-CMV-NIS-EGFP.pLVX-CMV-0-EGFP was constructed as control.BMSCs were isolated,cultured,and transfected by lentivirus.The antibiotic-resistant transfected BMSCs (BMSCs-NIS-EGFP and BMSCs-EGFP) were selected.The expression of NIS gene was examined by Western blot.Functional NIS activity was confirmed by the uptake of 125I and the inhibition effect of NaClO4.The nude mice intracranial glioma models were established.MicroSPECT was performed at 24 h post BMSCs-NIS-EGFP injection via the tail vein.Results pLVX-CMV-NIS-EGFP and pLVX-CMV-0-EGFP were successfully constructed and packaged.BMSCs were successfully isolated and cultured.Stable cell lines BMSCs-NIS-EGFP and BMSCs-EGFP were constructed after lentivirus transfection and puromycin selection.The expression of NIS gene was detected by Western blot in BMSCs-NIS-EGFP,but not in BMSCs-EGFP.BMSCs-NIS-EGFP showed significantly more uptake of 125I (nearly 10 times than the uptake in BMSCs-EGFP) and the uptake could be significantly inhibited by NaClO4.The nude mice intracranial glioma models were successfully established and the BMSCs-NIS-EGFP in glioma foci could be visualized by microSPECT imaging at 24 h post injection.Conclusions A recombinant lentivirus containing NIS gene could be successfully constructed for monitoring BMSCs migration towards intracranial glioma.It might provide evidence on the research of BMSCs and NIS gene mediated therapy for glioma.