VP22-CD级联增效基因工程化新型溶瘤病毒对脑胶质瘤干细胞疗效的研究

The limited median survival time of Glioblastoma was mainly because of the diffused infiltrative growth of glioma cells (GCs) and the chemotherapy and radiotherapy resistance to the residual glioma stem cells(GSCs) after synthesized surgical operation therapy, which calling for the imperative development of effective methods for glioblastoma treatment. This subject took advantage of the bystander effect of VP22, combined with the advantages of the cytosine deaminase / 5 - fluorocytosine suicide gene (CD/5-FC) system and the oncolytic virus(HSV-1), trying to reach the cascade amplified effect to glioma cells especially to gliomas stem cells and diffused infiltrative glioma cells. To obtain the glioma stem cells, human glioma specimens were primaryly cultured, identified by the cell surface marker CD133 and isolated by the flurescence activated cell sorting (FACS);Then glioma stem cells were cultured in conventional and three-dimensional cell culture, and the glioma stem cells' nude mouse model was established; VP22-CD gene was cloned into the oncolytic virus vector which deleted ICP47 gene to acheve the cascade amplified efficacy and targeted glioma stem cells more efficiently; Glioma therapy experiments were done individually at the conventional and the three-dimensional cell culture level in vitro and in the nude mouse model in vivo. Tumor size was observed and measured through the Magnetic Resonance Imaging (MRI) and the nude mouse's survival time of different therapy groups was tested; Tissue histopathology of glioma cells especially giloma stem cells and diffused infiltrative cells was analysed; The protein of newly constructed oncolytic virus and the proteins expressed in apoptosis and autophagy processes were measured by western blot. Consequently, our study clarifies the cascade amplified efficacy and the following mechanisms of newly constructed oncolytic virus to glioma cells especially glioma stem cells, trying to provide new method for glioma treatment.

恶性胶质瘤呈弥漫浸润性生长，且残存胶质瘤干细胞（GSCs）对化疗、放疗具有耐受性，其中位生存期极低，迫切需要寻找新的治疗手段。本课题利用VP22旁观者效应，结合胞嘧啶脱氨酶/5-氟胞嘧啶自杀基因系统（CD/5-FC）及溶瘤病毒(HSV-1)的双重优点，以期达到级联增效杀伤脑胶质瘤细胞尤其是胶质瘤干细胞的目的。通过人脑胶质瘤标本培养胶质瘤干细胞并鉴定后进行流式分选；常规及三维立体培养流式分选后胶质瘤干细胞并建立裸鼠脑胶质瘤干细胞模型；将VP22-CD基因插入到删除ICP47基因的溶瘤病毒（HSV-1）中，以对胶质瘤干细胞级联增效杀伤并更具靶向性；进行体内外胶质瘤干细胞的溶瘤病毒治疗实验，MRI观察肿瘤大小并分析荷瘤鼠生存时间；Western blot检测新构建溶瘤病毒蛋白表达并进行组织病理分析，从而明确新型溶瘤病毒对脑胶质瘤级联增效作用及其发生机制，为脑胶质瘤提供新的病毒-基因治疗方法。

本项目旨在研究VP22-CD溶瘤病毒对胶质瘤细胞，特别是胶质瘤干细胞的体内体外杀伤作用，为脑胶质瘤的治疗提供新的思路和方法。方法：1）体外实验：新鲜高级别（WHO分类Ⅲ、Ⅵ级）人脑胶质瘤标本经原代培养获得GSCs，免疫荧光技术检测未分化GSCs CD133、Nestin等的表达；单克隆形成实验检测GSCs的自我更新能力； 慢病毒感染胶质瘤细胞并流式分选获得稳定表达VP22-CD融合蛋白的胶质瘤细胞系 ；RT-PCR和Western blot检测VP22-CD融合蛋白的表达情况；MTT法检测5-FC前药及溶瘤病毒对稳定表达VP22-CD基因的胶质瘤细胞杀伤作用；2）体内实验：裸鼠颅内原位GSCs及携带VP22-CD融合蛋白肿瘤细胞移植瘤模型的构建；MRI观察5-FC前药及溶瘤病毒对荷瘤裸鼠肿瘤体积的影响，RT - PCR和Western blot及免疫组化检测VP22-CD融合蛋白的表达；最后观察病毒处理后荷瘤裸小鼠的生存情况。结果： 1）从高级别胶质瘤标本中培养出的GSCs，能够表达CD133及Nestin，并具有自我更新能力和多向分化能力； 2）成功获得稳定表达VP22-CD融合蛋白胶质瘤细胞系，检测显示细胞表达VP22-CD融合蛋白；3）前药5-FC及溶瘤病毒可显著抑制细胞生长（P<0.01），5-FC联合rHSV-1对胶质瘤细胞具有明显的联合抑制作用（P<0.01）；4）成功构建原位荷瘤小鼠模型，体内治疗中前药5-FC及rHSV-1可显著抑制颅内肿瘤细胞的生长（P<0.05），5-FC联合rHSV-1对颅内肿瘤具有明显的联合抑制作用（P<0.05）。5）western blot及免疫荧光检测显示肿瘤组织内具有VP22-CD融合蛋白的表达。结论：在VP22-CD融合蛋白稳定表达肿瘤细胞中，前药5-FC及rHSV-1对肿瘤具有明显的杀伤抑制作用；联合前药5-FC及rHSV-1对脑肿瘤细胞具有明显的协同作用；VP22-CD溶瘤病毒对脑胶质瘤治疗具有良好的应用前景，本课题为胶质瘤特异高效溶瘤病毒治疗打下良好的实验基础。