转染干扰素-γ的神经前体细胞治疗小鼠脑胶质瘤的实验研究

Immunogenetherapy of glioma by mIFN-γ transfected neural precursor cells: an experimental study

目的:探讨小鼠干扰素-γ(mIFN-γ)基因修饰胚胎干细胞诱导而来的神经前体细胞(NPC)用于脑胶质瘤基因治疗的可行性。方法:小鼠胶质母细胞瘤G422经NPCs/pTracer/mIFN-γ条件培养液培养后,MTT法体外观测其对瘤细胞的抑制效应,TUNEL法观测凋亡。DAPI标记瘤细胞,建立小鼠脑胶质瘤模型,NPCs/pTrac-er/mIFN-γ经立体定向方法注入小鼠颅内,荧光显微镜下观察肿瘤生长情况。结果:NPCs/pTracer/mIFN-γ条件培养液培养的胶质瘤细胞生长受到抑制,与对照组比较差异有统计学意义,χ2=7.21,P=0.032。以50%条件培养液处理24、48和72 h后每中倍视野凋亡细胞数分别为2.5±0.9、3.4±1.2和6.6±2.0,显著高于对照组,t值分别为2.37、2.56和2.80,P值分别为0.041、0.034和0.022。将基因修饰的NPC移植到小鼠脑肿瘤模型后,该细胞能向肿瘤细胞方向迁移,与肿瘤浸润范围一致并可追踪远地浸润的瘤细胞。第14天瘤体积测量,对照组和治疗组分别为(62.09±0.55)mm3和(27.54±0.86)mm3,治疗组显著小于对照组,t=4.16,P=0.015。NPC/pTracer/mIFN-γ治疗组平均生存时间为(28.5±0.98)d,较对照组延长,t=3.81,P=0.012。结论:IFN-γ基因修饰胚胎干细胞诱导而来的NPC可稳定表达外源基因,是脑胶质瘤基因治疗的良好载体。

OBJECTIVE： To examine the feasibility of using genetically modified embryonic stem （ES） cell-derived neural precursor cell （NPC） in glioma gene therapy. METHODS：After being cultured in NPCs/pTracer/mIFNq, conditioned medium, Cell proliferation of mouse malignant glioma cell line G422（s） was measured by MTT method and the apoptosis was observed by TUNEL in vitro. The mouse glloma model was established by using DAPI labeled G422 cell line. NPCs/pTracer/mIFN-γ was inoculated in intracranial glioma, then the distribution and migration of these cells in glioma-bearing brain were observed and the survival time and brain tumor volume were compared between inoculating NPCs/pTracer/mIFN-γ and the control. RESULTS： The was observed in the G422 cells incubated in the presence of NPCs/pTracer/mIFNq, conditioned medium （x^2： 7.21, P=0.032）. NPCs/pTracer/mIFN-γ conditioned medium induced G422 cells to apoptosis in vitro, after being treated with 50% conditioned medium 24 h, 48 h, 72 h later. The apoptic cell number was （2.5±0.9）/medium field, （3.4±1.2）/medium field and （6.6±2.0）/medium field, respectively, （t： 2.37,2. 56,2.80 respectively, P value： 0. 041, 0. 034, 0. 022 respectively）. With DAPI labeled G422 cells and GFP expressing NPCs, it demonstrated that NPCs, after being implanted into experimental intracranial gliomas in vivo, distributed themselves migrated uniquely in juxtaposition to widely expanding and aggressively advancing tumor cells. Glioma-bearing mice with IFN-γ-secreting NPCs demonstrated prolonged survival （28. 5±0.98） d, （P=0. 012） and the tumors were significantly smaller （27.54±0.86） mm^3 in size compared with those in the control groups （62. 09±0.55） mm^3 （P=0.015）. CONCLUSION： NPCs from genetically modified ES cells can be used as carriers for glioma gene therapy.