自杀基因治疗恶性胶质瘤的研究

Study on Suicide Gene Therapy of Malignant Glioma

目的：单纯疱疹病毒Ⅰ型胸苷激酶（ＨＳＶ－ｔｋ）基因治疗恶性胶质瘤体内外试验。方法：分子克隆及真核细胞基因转染技术构建逆转录病毒（ＲＶ）载体ｐＭＶ７（ｔｋ）及ＰＡ３１７ｔｋ包装细胞系；体外不同比例混合鼠Ｃ６胶质瘤细胞与ＰＡ３１７ｔｋ细胞，在ＧＣＶ（Ｇａｎｃｉｃｌｏｖｉｒ）作用下观察细胞存活率；建立ＳＤ大鼠颅内Ｃ６胶质瘤模型（种植５×１０５Ｃ６细胞），治疗组第３天原位注射５×１０６ＰＡ３１７ｔｋ细胞，５天后腹腔给予ＧＣＶ（３０ｍｇ／ｋｇ．ｄ），ＭＲＩ全程监测肿瘤消长，观察病症及存活期，并行病理检查。结果：在ＧＣＶ０．１～１０１μｇ／ｍｌ浓度范围内，Ｃ６细胞存活率随ＰＡ３１７ｔｋ细胞混入比例增加而逐渐减低（Ｐ＜０．００１），并具有ＧＣＶ剂量依赖性（Ｐ＜０．０１）；体内试验治疗组病症轻，生存期延长，ＭＲＩ表明治疗组肿瘤体积较对照组明显减小（Ｐ＜０．０１），１个月时病理检查见肿瘤细胞消失，代之以小胶质细胞增生并形成坏死囊。结论：应用本实验室构建的ＲＶ载体ｐＭＶ７（ｔｋ）及其ＰＡ３１７ｔｋ包装细胞系治疗恶性胶质瘤是一种有效及具有前途的治疗方法。

Objective: To study the effects of Herpes Simplex virus type 1 thymidine kinase (HSV-tk) gene and Ganciclovir (GCV)therapeutic system for malignant glioma in vitro and in vivo Methods: We constructed retrovirus (RV) vector bearing HSV-tk gene and established its packaging cell line PA317tk using the technique of molecular cloning and gene transfection for eukaryotic cells respectively; C6 glioma cells were mixed with PA317tk cells in various percentages and exposed to GCV in vitro, cell survival rate was determined with MTT method; C6 glioma model in vivo was established in SD rat (5105 C6 cells xenograft);5106 PA317tk cells were injected in situ on the third day after tumor cells xenograft GCV was administrated ip at the dose of 30mg/kg/day 5 days later Tumor growth or regression was monitored with MRI General manifestation after implantation with C6 glioma cells, survival time and pathological examination were also observed Results: Survival rate of C6 cells was negatively correlated with the percentage of PA317tk cells added (P< 0001), and its reduction was GCV dose dependent (P< 001) Milder symptomatic manifestation, longer survival and smaller tumor volume were obvious in the treatment group as compared to the control group Tumor cells almost disappeared and were replaced with the proliferation of glial cells and the formation of necrotic cyst Conclusion: The treatment of glioma using RV vector pMV7 (tk) and its packaging cell line PA317tk constructed and established in our laboratory is an effective and prospective modality of treatment