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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 verify the efficacy and safety of thymidine kinase (TK) gene-mediated hydroxymethyl acyclovir (GCV) system for gene therapy of human brain malignant gliomas. Methods The thymidine kinase (TKc) gene was isolated from the herpes simplex virus type I (HSV-I) strain (17) and DNA sequence analysis was carried out. A retrovirus recombinant vector (pLTKcSN) containing the TKc gene was constructed and Virus-producing cells containing the pLTKcSN virus (pLTKcSN/VPC). In vitro efficacy test: After co-culture with rat brain tumor C6 cells, pLTKcSN/VPC was treated with GCV. In vivo efficacy assay: Intracranially implanted C6 brain tumors were injected in situ with pLTKcSN/VPC. Safety Test: pLTKcSN/VPC was implanted into mice, rats and monkeys and GCV was injected to observe its toxic side effects. Results There were 5 nucleotide and 2 amino acid variations in the HSV-ITKc gene compared to the pOPFHSV-1TK gene. pLTKcSN and its pLTKcSN/VPC can effectively mediate cytotoxicity of GCV on C6 in vitro and in vivo. Its virus titer was 2 x 106 CFU/ml. After 48 mice, 24 rats and 6 monkeys, no severe toxic side effects and irreversible pathological changes were observed. Conclusion The thymidine kinase gene of the herpes virus is an effective and safe therapy for brain tumors. It can be used in clinical trials after drug trials.