红色荧光基因慢病毒转染人脑胶质瘤干细胞的实验研究

Labeling of stable glioma stem cells with red fluorescent protein transfected by lentiviral vector

目的建立体外稳定表达红色荧光蛋白（RFP）的人脑胶质瘤干细胞。方法将含有R印基因的慢病毒（RFP-lentivirus）转染体外培养的人脑胶质瘤干细胞SU2．荧光显微镜下观察红色荧光表达情况；流式细胞仪测定转染前SU2细胞、转染后IuP—SU2细胞传代培养20代后红色荧光转染率；动态观察IUP-SU2单细胞的分裂及克隆形成情况；免疫荧光染色检测RFP．SU2细胞CD133、nestin的表达。结果RFP-SU2呈悬浮球状生长，RFP呈高表达。转染前SU2细胞的红色荧光转染率仅为1．5％，转染并克隆培养20代后RFP—SU2细胞红色荧光转染率达75％。RFP—SU2单细胞仍可增殖形成脑肿瘤干细胞球，具有自我更新和克隆增殖能力。免疫荧光染色检测显示RFP．SU2细胞中CD133、nestin表达阳性。结论以RFP为生物标记物的人脑胶质瘤干细胞成功建立，可为将来人脑胶质瘤干细胞的深入研究提供理想的材料。

Objective To establish stable glioma stem cells with a high expression level of red fluorescent protein （KFP） in vitro. Methods The glioma stem cells SU2 were transfected with lentivirus vector containing IFP gene; cell expression of RFP was observed by fluorescent microscopy; RFP-positive glioma stem cells were sorted out by fluorescence-activated cell sort. The transfection efficiency of SU2 cell before transfection and 20-passaged RFP-SU2 cells after tmnsfection were assayed by flow cytometry. Dynamic viewing was performed to observe the cell division and cloning of RFP-SU2 single cell; RFP -positive cells were collected and immunostained with antibodies against CD133 and nestin. Results RFP-SU2 cells grew as levitated sphere with high RFP expression; the transfection efficiency of SU2 cell before transfection was only 1.5%, while that of 20-passaged RFP-SU2 cells after transfection reached to 75%. RFP-SU2 single cell could proliferate into brain tumor stem cell spheres, having the abilities of self-renewing and clonal proliferation. Immunofluorescence showed positive- CD133 and nestin expressions in RFP-SU2 cells. Conclusion A SU2/RFP cell line marked by RFP is established; and it can serve as a promising tool for further basic research.