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目的:本研究以端粒酶逆转录酶(humantelomerasereversetrascriptase,hTERT)基因的全长序列构建反义hTERT的真核表达载体,以此来转染肿瘤细胞PLA 801D,观察反义hTERT对其细胞周期分布及对p53表达的影响。方法:构建了包含hTERT全长基因序列的反义hTERT基因真核表达载体pcDNA3. 1(-) -hTERT,并稳定转染人肺巨细胞癌细胞系PLA 801D,观察基因转染前后,流式细胞术检测PLA 801D细胞的周期分布,用半定量RT- PCR检测p53mRNA的表达水平,免疫组化检测突变型p53蛋白阳性表达率的差异。结果:全长的反义hTERT基因可显著地抑制p53mRNA的表达,使细胞周期的分布出现G0 /G1期阻滞,突变型p53蛋白阳性率显著下降。结论:反义的hTERT基因可有效的抑制p53mRNA及突变型蛋白的表达水平,引起细胞周期阻滞,使细胞的恶性增殖减缓。
OBJECTIVE: To construct the eukaryotic expression vector of antisense hTERT by using the full-length sequence of human telomerase reverse transcriptase (hTERT) gene in order to transfect tumor cell PLA 801D and observe its cell cycle distribution And its effect on p53 expression. Methods: The antisense hTERT gene eukaryotic expression vector pcDNA3.1 (-) - hTERT was constructed and transfected into human lung giant cell carcinoma cell line PLA 801D stably. The cell cycle distribution of PLA 801D cells was detected by cytology. The expression of p53 mRNA was detected by semi-quantitative RT-PCR. The difference of the positive expression rate of the mutant p53 protein was detected by immunohistochemistry. RESULTS: The full-length antisense hTERT gene could significantly inhibit the expression of p53 mRNA and block the G0 / G1 phase of the cell cycle. The positive rate of mutant p53 protein was significantly decreased. Conclusion: Antisense hTERT gene can effectively inhibit the expression of p53 mRNA and mutant protein, causing cell cycle arrest and slowing the malignant proliferation of cells.