脑肿瘤二级康复预防:阳离子脂质体介导HSV-TK/ACV系统对人脑胶质瘤细胞增殖活性的影响

背景基因治疗是国内外对于脑胶质瘤生物学治疗的研究热点.目的应用已克隆并构建的真核表达载体pCR3-TK,探讨HSV-TK/ACV系统对人脑胶质瘤细胞抑制生长和杀伤作用研究.设计以细胞为研究对象的实验研究.单位一所大学医院神经外科、肿瘤内科.对象实验于2004-01/04在哈尔滨兽医研究所生物技术国家重点实验室完成.所用的真核表达载体pCR3-TK由作者构建,TJ905细胞株由天津神经病学研究所浦佩玉教授惠赠.选择未转染和转染空载体的细胞作为对照组.方法用阳离子脂质体Lipofectamine将pCR3-Uni及含HSV-TK基因的真核表达载体pCR3-TK转染至人脑胶质瘤细胞株TJ905中,筛选出阳性克隆,阳性细胞克隆给予ACV(50 mg/L),72 h后,收集玻片,进行AgNOR染色.主要观察指标对未转染和转染不同载体的TJ905细胞ACV作用后银染颗粒进行记数.结果转染HSV-TK基因的细胞,在给予ACV后,细胞增殖活性明显降低,转染pCR3-Uni和pCR3-TK细胞克隆的AgNOR颗粒数分别为14.33和6.67(P＜0.01).结论AgNOR计数是一种操作简便、检测细胞增殖活性的方法,为研究HSV-TK/ACV系统的抗肿瘤机制提供帮助.

Phase Ⅱ rehabilitation/prevention of brain tumor: effect of the HSV-TK/ACV system mediated by cationic lipsome on the proliferative activity of human glioma cells

BACKGROUND: Gene therapy is a popular topic in domestic and overseas studies on biological therapy for brain tumor.OBJECTIVE: By using a newly constructed eukaryotic expression vector of pCR3-TK, the effect of the HSV-TK/ACV system on the proliferative activity of human glioma cells was investigated.DESIGN: Experimental study based on cells.SETTING: Department of neurosurgery and department of oncology in a university hospital.MATERIALS: The study was conducted at the National Key Laboratory of Veterinary Biotechnology of Harbin Veterinary Research Institute from January to April in 2004. The eukaryotic expression vector of pCR3-TK was constructed by the author. The TJ905 strain was a gift from professor Pu Pei-yu, who worked in the Neurology Institute of Tianjin city. The nontransfected cells and the cells transfected with pCR3-Uni vector were set as controls.METHODS: By using Lipofectamine(a cationic liposome), the pCR3-Uni vector and the recombinant pCR3-TK plasmid(inserted with HSV-TK gene)were transfected into the human glioma cell strain-TJ905. Then the positive clones were picked out and were given ACV(50 mg/L) . Totally 72 hours later, the cover slips were collected and silver staining for nucleolus organizer regions(AgNORs) was performed.MAIN OUTCOME MEASURES: After the ACV treatment and AgNORs staining, the numbers of silver-stained granules in TJ905 cells with or without transfections were counted respectively.RESULTS: In those cells transfected with HSV-TK gene, after ACV treatment, a significant decreasing in proliferative activity could be observed, and the average numbers of the silver-stained granules in cells transfected with pCR3-Uni or pCR3-TK were 14.33 and 6.67 respectively( P ＜ 0.01).CONCLUSION: As an easy-to-operate method, AgNOR counting is helpful for the studies on the proliferative activity of cells and the investigations into the potential anti-tumor mechanism of the HSV-TK/ACV system.