内皮抑素和血管生成抑素融合基因修饰的单纯疱疹病毒对胶质瘤干细胞的体外实验研究

Efficacy of genetically engineered oncolytic virus on glioma stem cells in vitro

目的探讨内皮抑素和血管生成抑素（Endo—Angio）融合基因修饰的单纯疱疹病毒（VAE）对胶质瘤干细胞（GSCs）的体外溶瘤作用。方法新鲜高级别（WHOⅢ级、Ⅵ级）人脑胶质瘤标本经原代培养获得GSCs，采用流式细胞术检测其中CD133阳性细胞数，单克隆形成实验检测GSCs的自我更新能力，免疫荧光技术检测未分化GSCs的Nestin及分化后GFAP、NF和MAG的表达；MTS法检测VAE对GSCs增殖活性的影响，RT—PCR和Western blot检测Endo—Angio融合蛋白的表达并检测其对人脑微血管内皮细胞（HBMEC）增殖的影响；最后观察病毒处理后仍存活细胞的自我更新和诱导贴壁情况。结果（1）从20例高级别胶质瘤标本中培养出4例GSCs，能够表达CD133及Nestin，具有自我更新能力和多向分化能力。（2）VAE能够感染GSCs，4例GSCs增殖活性明显下降（P〈0．05）。（3）VAE感染GSCs 48h后，基因水平及蛋白水平都有Endo—Angio融合蛋白表达，该融合蛋白使HBMEC增殖活性明显下降（P〈0．05）。（4）VAE感染后仍存活的细胞不再具有形成GSCs球的能力，加入血清诱导后也不再贴壁生长。结论在体外，VAE能够显著抑制GSCs活性，能够表达具有生物活性的Endo—Angio融合蛋白，为脑胶质瘤溶瘤病毒治疗开辟了新的途径。

Objective Exploring the effect of endostatin and angiostatin （Endo -Angio） fusion gene - modified herpes simplex virus （VAE） on glioma stem cells （ GSCs ） in vitro. Method Surgical specimens of human high grade glioma （ WHO Ⅲ, Ⅳ） were collected, and GSCs were isolated under the culture conditions originally designed for selective expansion of neural stem cells. In order to identify GSCs ,the number of CD133 positive cells in GSCs were detected by flow cytometry ; self - renewal capacity and the expression of Nestin, GFAP, NF, and MAC, of GSCs were detected by monoclanal forming and immunofluorescence assay respectively. After that, GSCs viability was detected by MTS assay. Expression of Endo - Anglo fusion gene at mRNA and protein level was detected by RT - PCR and Western blot. At last, the efficacy of fusion protein to human brain micruvascular endothelial cells （HBMEC） and the biological properties of GSCs were detect after infected by VAE. Results （1）4 GSCs isolated from 20 surgical specimens could suspend growth and had the ability of self- renewal and multipotential differentiation,and could express neural stem cells marker,CD133 and Nestin. （2）VAE could infect GSCs and significantly inhibit the viability of GSCs （ P 〈 0. 05 ）. （3）Significant expression of Endo- Angio fusion gene was observed and it could inhibit HBMEC proliferation （ P 〈 0. 05 ）. （4） Residual viable cells lost the ability of self-renewal and adherent differentiation. Conclusions In vitro, VAE can inhibit the activity of GSCs and the expression of exogenous Endo - Angio fusion gene can inhibit HBMEC proliferation. VAE can be used as a novel virus -gene therapy strategy for glioma.