TTDR双调控溶瘤性HSV-1工程化间充质干细胞治疗脑胶质瘤研究

The limited survival time of glioblastoma is mainly because of the diffused growth character of glioma cells (GCs) and the resistance of the glioma stem cells(GSCs) to the radiotherapy/chemotherapy after surgical operation,which calling for the imperative development of effective methods for glioblastoma treatment.This subject aims to use the transcription and translation dual-regulated(TTDR) HSV-1 to infect and lyse the glioma stem cells specifically and efficiently.Tumor microenvironment will be modulated and the virus dissemination will be faciliated by the immune factor inserted in the oncolytic virus. Messenchymal stromal cells are used to enhace the viability of the oncolytic viruses and carry them to the tumor sites.Human glioma stem cells were primarily cultured, identified by the cell surface marker CD133 and isolated through the flurescence activated cell sorting (FACS). Cells will be cultured either in conventional or the three-dimensional cell culture way. Nude mouse and rat in situ therapy model of GCs or GSCs will also be established. Theraputic experiments of oncolytic virus(or the MSCs infected with it) to the GCs or GSCs will be done in vitro and in vivo respectively. Tumor size will be observed and measured through the Magnetic Resonance Imaging (MRI) and the animal survival times of different therapy groups will be tested. Tissue histopathology of GCs or GSCs, proteins expression and immune factors will be analysed. Taken together, our study aims to clarify the specific efficacy of TTDR-HSV-1 (or the MSCs infected with it) in the inhibition of GCs or GSCs, trying to provide a new method for glioma therapy.

恶性胶质瘤呈弥漫浸润性生长，术后易复发，残存的胶质瘤干细胞对放化疗不敏感，迫切需要探寻新的治疗策略。本研究利用转录及翻译双调控溶瘤病毒（TTDR-oHSV-1）高效、靶向杀伤胶质瘤干细胞；利用自身携带的免疫因子调节肿瘤微环境，促进病毒的扩散；并利用间充质干细胞（ MSCs）作为细胞载体延长病毒的存活时间并向胶质瘤细胞富集、迁移的特性，以期协同达到高效、精准杀伤肿瘤细胞目的。通过脑胶质瘤标本完成胶质瘤干细胞培养、鉴定、流式分选；常规及三维立体培养细胞并建立裸鼠及大鼠脑胶质瘤模型；分别对溶瘤病毒及病毒工程化的MSCs进行体内外治疗实验，观察溶瘤病毒对肿瘤的杀伤作用、 肿瘤微环境的变化、体内外免疫因子及蛋白表达的变化。利用MRI及小动物活体成像仪观察肿瘤体积并分析荷瘤鼠生存时间，HE染色及免疫荧光分析组织病理变化，明确TTDR-oHSV-1治疗脑胶质瘤作用及其机制，为脑胶质瘤提供新的治疗方法。

胶质母细胞瘤是一种具有免疫抑制、高血管密度和破坏性的恶性脑肿瘤。即使采用包括手术、放疗、化疗在内的联合治疗，胶质母细胞瘤患者的预后仍然非常差。溶瘤腺病毒可以特异性地在胶质母细胞瘤中复制，允许其携带的治疗性基因快速复制。此外，E1A是腺病毒复制的必须基因，是病毒感染肿瘤细胞后第一个表达的基因。E1A的表达可由Ki67启动子调控，而CMV启动子驱动治疗性基因的表达。然而，由Ki67核心启动子驱动并携带IL-15的双调控溶瘤腺病毒对胶质母细胞瘤的疗效尚未被研究。因此，我们成功构建了一种由Ki67核心启动子驱动并携带IL-15的双调控溶瘤腺病毒，该病毒能够选择性感染并杀死胶质母细胞瘤，同时几乎不损害正常细胞。此外，溶瘤腺病毒不仅直接抑制血管生成，而且通过减少VEGF的分泌表现出强大的抗血管生成能力，而且IL-15能够通过改善肿瘤微环境，进而展现出强大的抗肿瘤潜力，并能够延长动物的生存期。这些结果为Ki67核心启动子驱动并携带IL-15的双调控溶瘤腺病毒在胶质母细胞瘤治疗中的作用提供了新的视角，这可能有助于开发实体肿瘤的新疗法。