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目的研究端粒酶逆转录酶(hTERT)与c-myc基因反义寡核苷酸(ASODN)对HL-60细胞端粒酶活性的影响,探讨HL-60细胞端粒酶活性与hTERT和c-myc基因表达的关系。方法应用反义寡核苷酸(ASODN)分别封闭HL-60细胞hTRET与c-myc基因,RT-PCR方法检测基因表达;分别使用TRAP-ELISA法、PAGE-银染法检测细胞端粒酶活性。结果ASODN作用细胞72 h后,hTERT、c-mycmRNA的表达受到不同程度抑制,以30μmol/L作用组表达量最低。TRAP-ELISA检测:hTERT ASODN 10、20、30μmol/L作用组,c-mycASODN 20、30μmol/L作用组与未作用组比较,端粒酶活性明显降低(P<0.05);c-mycASODN 10μmol/L作用组及c-myc、hTERT SODN作用组与未作用组比较无显著差异(P>0.05)。PCR-PAGE检测:hTERT与c-mycASODN作用组较SODN作用组端粒酶条带明显减少,以30μmol/L作用组条带最少,而同浓度hTERT ASODN作用组较c-mycASODN作用组条带减少。结论hTERT与c-myc基因ASODN能够分别抑制该基因mRNA的表达,同时具有下调端粒酶活性作用。
Objective To study the effect of telomerase reverse transcriptase (hTERT) and c-myc antisense oligonucleotide (ASODN) on the telomerase activity of HL-60 cells and to explore the relationship between the telomerase activity of HL-60 cells and hTERT and c -myc gene expression. Methods Antisense oligonucleotide (ASODN) was used to block the hTRET and c-myc gene of HL-60 cells respectively. The gene expression was detected by RT-PCR. The telomerase activity was detected by TRAP-ELISA and PAGE- . Results The expression of hTERT and c-myc mRNA was inhibited to some extent in ASODN-treated cells for 72 h, and the expression was lowest in 30 μmol / L group. The results of TRAP-ELISA showed that telomerase activity was significantly decreased in hTERT ASODN 10, 20 and 30μmol / L groups (P <0.05), while c-myc ASODN 20 and 30μmol / L group and c-myc and hTERT SODN groups had no significant difference (P> 0.05). The results of PCR-PAGE showed that telomerase band of hTERT and c-mycASODN group was significantly decreased compared with that of SODN group, the band of 30μmol / L group was the lowest, while the band of hTERT ASODN group was lower than that of c-mycASODN group . Conclusion hTERT and c-myc gene ASODN can inhibit the mRNA expression of this gene, meanwhile, it can down-regulate telomerase activity.