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目的:观察 c-myc 基因特异性核酶对肝癌细胞增殖的抑制作用。方法:计算机辅助设计并合成能特异性切割c-myc mRNA 的锤头状核酶基因,定向克隆于逆转录病毒载体 pDOR。用脂质体(lipofectin)将重组载体 pDOR-RZ 转染HCC9204细胞,G418筛选出阳性细胞。原位杂交和斑点杂交检测核酶在细胞内的表达。用 ABC-ELISA,流式细胞仪和MTT 分别检测转核酶基因肝癌细胞 c-myc 蛋白表达,细胞周期及细胞增殖的改变。电镜观察形态学改变。裸鼠体内致瘤性试验。结果:原位及斑点杂交显示核酶已在肝癌细胞内成功表达。转核酶基因细胞 c-myc 蛋白表达受到抑制。细胞周期显示 G_1期细胞增多,S 期细胞减少。MTT 试验显示细胞增殖能力下降。电镜发现细胞空泡变性,染色质凝集成块状。裸鼠体内致瘤率降低,移植瘤生长速度减慢。结论:核酶技术为肿瘤基因治疗提供了一种特异性的新手段。
Objective: To observe the inhibitory effect of c-myc gene-specific ribozymes on the proliferation of hepatocellular carcinoma cells. METHODS: Computer-aided design and synthesis of hammerhead ribozyme genes that specifically cleave c-myc mRNA were targeted for cloning in the retroviral vector pDOR. The recombinant vector pDOR-RZ was transfected into HCC9204 cells with lipofectin, and positive cells were selected by G418. In situ hybridization and dot blot hybridization were used to detect the intracellular expression of ribozymes. The expression of c-myc protein, cell cycle and cell proliferation were detected by ABC-ELISA, flow cytometry and MTT, respectively. Electron microscopy to observe morphological changes. In vivo tumorigenicity test in nude mice. Results: In situ and dot blot hybridization showed that the ribozyme has been successfully expressed in hepatoma cells. Transfer of ribozyme gene cell c-myc protein expression was inhibited. The cell cycle showed an increase in cells in the G1 phase and a decrease in cells in the S phase. MTT assay showed a decrease in cell proliferation. Electron microscopy revealed vacuolar degeneration of the cells, and chromatin aggregated into clumps. The tumorigenicity in nude mice was reduced, and the growth rate of transplanted tumors was slowed down. Conclusion: Ribozyme technology provides a specific new method for tumor gene therapy.