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目的探讨人端粒酶催化亚基(hTERT)反义cDNA(pAdeasy-hTERT)在体外对胶质瘤细胞生长的抑制作用。方法将腺病毒介导的pAdeasy-hTERT作用于人胶质瘤细胞系U251,用MTT法检测细胞存活率、端粒酶重复序列扩增法测定端粒酶活性、Westem杂交鉴定hTERT蛋白的表达、RT-PCR法检测hTERT cDNA水平、PCNA观察肿瘤细胞的凋亡情况以及用流式细胞仪对转染后肿瘤细胞的周期进行分析。结果pAdeasy-hTERT在体外明显抑制肿瘤细胞生长,降低端粒酶活性,抑制hTERT表达。结论pAdeasy-hTERT显著抑制人胶质瘤细胞生长,可成为恶性胶质瘤基因治疗的靶基因。
Objective To investigate the inhibitory effect of human telomerase catalytic subunit (hTERT) antisense cDNA (pAdeasy-hTERT) on glioma cell growth in vitro. Methods Adenovirus-mediated pAdeasy-hTERT was applied to human glioma cell line U251. The cell viability was detected by MTT assay. The telomerase activity was determined by telomerase repeat amplification assay. The expression of hTERT protein was identified by Westem hybridization. The hTERT cDNA level was detected by RT-PCR, the apoptosis of tumor cells was observed by PCNA, and the cycle of tumor cells was analyzed by flow cytometry. Results pAdeasy-hTERT significantly inhibited tumor cell growth in vitro, decreased telomerase activity and inhibited hTERT expression. Conclusion pAdeasy-hTERT significantly inhibits the growth of human glioma cells and may be the target gene of malignant glioma gene therapy.