CAJ | 학술논문

目的探讨双报告基因分别标记肝癌细胞、骨髓间充质干细胞(hMSCs),并利用活体成像技术动态监测肝癌细胞及骨髓间充质干细胞的可行性.方法分别构建同时表达荧光蛋白及荧光素酶报告基因的慢病毒载体,制备慢病毒,获得增强绿色荧光蛋白(eGFP)表达阳性的Hepa1-6小鼠肝癌细胞以及mKate2表达阳性的hMSCs;应用生物发光成像在细胞及活体水平评价其发光能力;Transwell实验探讨hMSCs向Hepa1-6小鼠肝癌细胞的迁移能力.结果成功构建慢病毒载体,并获得eGFP+-Hepa1-6及mKate2+-hMSCs细胞系;荧光显微镜观察细胞eGFP、mKate2表达,生物发光成像观察细胞荧光素酶表达光信号,其信号强度与细胞数目呈正相关性(R2分别为0.999和0.984);Transwell实验观察到hMSCs向肝癌细胞趋化.结论本研究开发构建适合荧光/生物发光双功能显像的慢病毒载体,成功转染Hepa1-6及hMSCs,为随后活体动态监测间充质干细胞对肿瘤生长情况的影响提供了可行性;并与体外实验相互印证,为研究骨髓间充质干细胞在肝癌发生发展中的作用提供了依据.
목적 탐토쌍보고기인분별표기간암세포、골수간충질간세포(hMSCs),병이용활체성상기술동태감측간암세포급골수간충질간세포적가행성.방법 분별구건동시표체형광단백급형광소매보고기인적만병독재체,제비만병독,획득증강록색형광단백(eGFP)표체양성적Hepa1-6소서간암세포이급mKate2표체양성적hMSCs;응용생물발광성상재세포급활체수평평개기발광능력;Transwell실험탐토hMSCs향Hepa1-6소서간암세포적천이능력.결과 성공구건만병독재체,병획득eGFP+-Hepa1-6급mKate2+-hMSCs세포계;형광현미경관찰세포eGFP、mKate2표체,생물발광성상관찰세포형광소매표체광신호,기신호강도여세포수목정정상관성(R2분별위0.999화0.984);Transwell실험관찰도hMSCs향간암세포추화.결론 본연구개발구건괄합형광/생물발광쌍공능현상적만병독재체,성공전염Hepa1-6급hMSCs,위수후활체동태감측간충질간세포대종류생장정황적영향제공료가행성;병여체외실험상호인증,위연구골수간충질간세포재간암발생발전중적작용제공료의거.
Objective To establish murine liver cancer cell line (Hepa1-6) and human mesenchymal stem cells (hMSCs) with dual-modality imaging lentiviral vector transfection and monitor the development of liver tumors and dynamically tracking of hMSCs by bioluminescent imaging in vivo.Methods The cell lines of Hepa 1-6 and hMSCs were constructed abd tranfected with RL-eGFP and FL2-mKate2 lentiviruses respectively.Then the eGFP+-Hepa1-6 and mKate2+-hMSCs cell lines were selected by fluorescence activated cell sorting (FACS).The bioluminescent imaging technology was employed to evaluate their bioluminescent abilities in vitro and in vivo.And the capacity of mKate2 +-hMSCs migrating to HCC cell line (eGFP +-Hepa1-6) was evaluated by Transwell migration assay.Results DNA sequencing analysis confirmed that gene sequences of lentiviral vectors were correct without mutation.We successfully constructed the lentivirus vector,produced lentivirus and infected Hepa1-6 and hMSCs respectively.Subsequently eGFP+-Hepa1-6 cells and mKate2+-hMSCs were obtained by FACS.The expressions of fluorescent protein eGFP and mKate were confirmed by fluorescence microscope.By bioluminescent imaging system,bioluminescence intensity strongly correlated with cell number.The Transwell assay showed that hMSCs migrated toward heptocellular carcinoma (HCC) cells.Conclusion A lentivirus vector has been successfully constructed for infecting Hepa1-6 and hMSCs and analyzed by fluorescence and bioluminescent imaging.And that makes it technically feasible to further monitor the interaction of hMSCs and liver tumor cells.These results provide rationales for further studying the mechanism of MSCs in the development and progression of HCC.