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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(50mg/L),72h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。
Background: Gene therapy is a hot research topic in the biological treatment of glioma at home and abroad. OBJECTIVE: To investigate the inhibitory effect of HSV-TK / ACV system on human glioma cell growth and killing by using the eukaryotic expression vector pCR3-TK cloned and constructed. Design: Experimental study of cells as a research object. Unit: a University Hospital neurosurgery, oncology. PARTICIPANTS: The experiment was performed at the State Key Laboratory of Biotechnology, Harbin Veterinary Research Institute from January to April 2004. The eukaryotic expression vector pCR3-TK used was constructed by the author, and the TJ905 cell line was kindly donated by Professor Pu Peiyu from Tianjin Neurological Institute. Cells that were not transfected and transfected with empty vector were selected as control group. Methods: The eukaryotic expression vector pCR3-Uni containing pCR3-Uni and HSV-TK gene was transfected into human glioma cell line TJ905 with cationic liposome Lipofectamine. The positive clones were screened out and the positive clones were given ACV ( 50mg / L). After 72h, the slides were collected and stained with AgNOR. MAIN OUTCOME MEASURES: Silver-stained particles were counted after ACV treatment on TJ905 cells transfected with different vectors. Results: The proliferation of HSV-TK transfected cells was significantly decreased after ACV administration. The number of AgNOR particles transfected into pCR3-Uni and pCR3-TK cells was 14.33 and 6.67, respectively (P <0.01). Conclusion: AgNOR counting is a simple and convenient method to detect cell proliferation activity, which can be used to study the antitumor mechanism of HSV-TK / ACV system.