反义hTERT对人肺巨细胞癌细胞系p53表达的影响

Antisense human telomerase reverse transcriptase repress the expression of p53 of human pulmonary giant cell carcinoma cell line

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:In this study, the authors investigated the effects of the full length of antisense human telomerase reverse transcriptase (hTERT) gene on the expression of p53 and the distrubution of cell cycles of human pulmonary giant cell carcinoma cell line (PLA-801D). Methods:An antisense hTERT gene eukaryotic expression vector pcDNA 3.1(-)-hTERT including the full-length region of hTERT CDS was constructed by recombinant DNA technique and transfected into the human pulmonary giant cell carcinoma cell line (PLA-801D). Then the expression of p53 mRNA was observed with RT-PCR, distrubution of cell cycles was determined by flow cytometry. The expression of mutated p53 protein was tested by immunohischemitry. Results:Full length (3.4kb) of hTERT fragment was inserted into vector pcDNA 3.1(-) in reverse orientation have been constructed and was transfected successfully into the PLA-801D cells, the distribution of cell cylce of antisense hTERT transfected PLA-801D cells was changed, and the antisense hTERT gene can act as an agent for specific inhibiting to p53 mRNA and the mutated p53 protein expression. Conclusion:The results suggest that full-length antisense hTERT can directed suppressed p53 mRNA expression level and down regulated mutated p53 protein.