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目的探讨合成端粒酶逆转录酶(hTERT)基因及端粒酶RNA(hTR)的小干扰RNA(small interfering RNA,siRNA)对皮肤T细胞淋巴瘤(CTCL)细胞株Hut78端粒酶活性的影响。方法采用体外转录法分别合成两条hTERT—siRNA及hTR—siRNA。采用siRNA直接处理细胞裂解液及磷酸钙共沉淀法将siRNA转染入Hut78细胞两种处理方式,应用端粒酶重复序列扩增聚合酶链反应(TRAP—PCR) 及聚丙烯酰胺凝胶电泳银染检测端粒酶活性的变化。结果体外转录法能高效合成siRNA,产量达 22．4 μg／40μL siRNA合成体系。经250 ng siRNA直接处理细胞裂解液后,可高效降低端粒酶的活性,抑制率达87％左右。30 ng siRNA转染细胞36 h后,可降低端粒酶活性20％左右,而250 ng siRNA可降低 75％左右。结论体外转录法能高效、快速、大量合成siRNA,针对hTERT及hTR基因的siRNA可明显降低Hut78细胞株的端粒酶活性。
Objective To investigate the effect of telomerase reverse transcriptase (hTERT) gene and telomerase RNA (hTR) small interfering RNA (siRNA) on the telomerase activity of human cutaneous T cell lymphoma (CTCL) cell line Hut78 . Methods Two hTERT-siRNA and hTR-siRNA were synthesized by in vitro transcription. SiRNA was used to directly process the cell lysate and calcium phosphate co-precipitation method to transfect siRNA into Hut78 cells. TRAP-PCR and polyacrylamide gel electrophoresis Dye detection of changes in telomerase activity. Results The in vitro transcription method could efficiently synthesize siRNA with the yield of 22.4 μg / 40μL siRNA synthesis system. After 250 ng siRNA direct treatment of cell lysate, the activity of telomerase can be reduced efficiently with the inhibition rate of about 87%. After 30 ng of siRNA transfected cells for 36 h, the telomerase activity was reduced by about 20%, while 250 ng siRNA was reduced by about 75%. Conclusion In vitro transcription can efficiently and rapidly synthesize siRNA in large quantities. SiRNA targeting hTERT and hTR genes can significantly reduce telomerase activity in Hut78 cells.