hTERT反义cDNA对人脑胶质瘤细胞的抑制作用

Inhibitory effects of antisense hTERT cDNA on proliferation of human glioma cells

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 effects of human telomerase reverse transcriptase （hTERT） antisense cDNA on human glioma cell proliferation in vitro. Methods We transfected antisense hTERT cDNA into human glioma U251 cells with adenovirus. The cellular viability of human glioma cells was analyzed by MTT assay. The telomerase activity was determined by telomeric-repeat amplification protocol enzyme-linked immunosorbent assay （TRAP-ELISA）. The expression of hTERT protein was examined by Western blot. The expression of hTERT cDNA was measured by reverse transcription polymerase chain reaction （RT-PCR）. The apoptosis of cells was investigated by proliferating cell nuclear antigen （PCNA） expression. The cell cycle distribution was assayed by flow cytometry. Results The growth of glioma cells transfected with antisense hTERT cDNA was significantly inhibited compared with the control cells; the telomerase activity was obviously down-regnlated; the expression of hTERT cDNA was distinctly restrained. Conclusion The growth of glioma cells transfected with antisense hTERT was markedly inhibited, indicating hTERT is a potential target for glioma gene therapy.