疱疹病毒I型中国株胸苷激酶基因治疗脑瘤的临床前研究  

Preclimical studies on thymidine kinase gene(TK)and gancyclovir system for treatment of malignant astrocytoma

作　　者：许秀兰[1,2] 张晓鹏[1,2] 郭忠[1,2] 胡加飞 朱诚 顾健人[1,2] 

机构地区：[1]上海市肿瘤研究所癌基因及相关基因国家重点实验室 [2]第二军医大学附属长征医院

出　　处：《中华医学杂志》1997年第1期35-38,共4页National Medical Journal of China

基　　金："八六三"生物高技术领域基金

摘　　要：目的验证胸苷激酶（ＴＫ）基因介导的羟甲基无环鸟苷（ＧＣＶ）系统对人脑恶性神经胶质瘤基因治疗的有效性及安全性。方法从中国疱疹病毒Ｉ型（ＨＳＶ－Ｉ）株（１７）中分离了胸苷激酶（ＴＫｃ）基因，并作了ＤＮＡ序列分析，组建了含ＴＫｃ基因的逆转录病毒重组载体（ｐＬＴＫｃＳＮ）及其含ｐＬＴＫｃＳＮ病毒的病毒生产细胞（ｐＬＴＫｃＳＮ／ＶＰＣ）。体外有效性试验：应用ｐＬＴＫｃＳＮ／ＶＰＣ与大鼠脑瘤Ｃ６细胞共培养后用ＧＣＶ处理。体内有效性试验：以ｐＬＴＫｃＳＮ／ＶＰＣ原位注入大鼠颅内移植的Ｃ６脑瘤。安全性试验：通过小鼠、大鼠和猴植入ｐＬＴＫｃＳＮ／ＶＰＣ并注射ＧＣＶ观察其毒副作用。结果ＨＳＶ－ＩＴＫｃ基因与ｐＯＰＦＨＳＶ－１ＴＫ基因比较有５个核苷酸和２个氨基酸的变异。ｐＬＴＫｃＳＮ及其ｐＬＴＫｃＳＮ／ＶＰＣ，在体内外能有效地介导ＧＣＶ对Ｃ６产生细胞毒作用。其病毒滴度为２×１０６ＣＦＵ／ｍｌ。经过４８只小鼠、２４只大鼠和６只猴体内未见严重毒副作用和不可逆的病理改变。结论疮疹病毒胸苷激酶基因治疗脑肿瘤是有效和安全的，通过药审后可用于临床试验。Objective To study preclinically TK gene mediated GCV system for treatment of human malignant astrocytoma.Methods The TK gene(TKc)was isolated from a Chinese strain of HSV I(17) and sequenced.Aretroviral vector with TKc(pLTKcSN) and its package cell line(pLTKcSN/VPC) were constructed.The in vitro assay for its activity was performed by mixed cultures of TK producer cells and rat glioma C 6 cells after treatment with GCV.The in vivo efficacy was examined by in situ inoculation of virus producer cells after intracerebral implantation of C 6 cells.Safety tests were analyzed by inoculation of pLTKcSN/VPC and injection of GCV,in mouse,rat,and Rhesus monkey,evaluated by histopathological examination and in situ hybridization with TK probe.Results TKc gene contained 5 nucleotide difference covering 2 amino acid variation.The retroviral vector pLTKcSN and its producer cell pLTKcSN/VPC(with atiter at 0.5 1.0×10 6 CFU/ml)can efficiently mediate cytotoxicity of GCV to C 6 cells,both in vitro and in vivo.The above system had no serious adverse effect and remarkable or irreversible pathological changes in48mice,24 rats and 6 Rhesus monkeys.Conclusion The preclinical studies indicated that it would be effective and safe for clinical trial after approval.

关 键 词：疱疹病毒 脑肿瘤 基因治疗 胸苷激酶 

分 类 号：R739.410.5[医药卫生—肿瘤] R730.59[医药卫生—临床医学]