RNA干扰沉默hTERT基因对大肠癌SW480细胞生物学特性的影响

An experimental study on colorectal carcinoma cell biological characteristics by RNA interferencetechnology of human telomerase reverse transcriptase gene silence

目的观察人端粒酶催化亚单位（hTERT）基因对SW480细胞生物学特性的影响方法将重组质粒pGPU6／GFP／Neo—hTERT—shRNA（hTERT—shRNA）用脂质体转染法导人大肠癌自胞株SW480，实验分4组，每组设3个复孔，分别于转染后24、48、72h，逆转录一聚合酶链反压（RT．PCR）检测hTERTmRNA的表达，免疫细胞化学法检测hTERT蛋白表达，PCR—TRAP-ELISA{；检测端粒酶活性，流式细胞仪检测细胞周期分布，终端脱氧核苷酸转移酶介导的缺口末端标自（TUNEL）法和透射电镜观察细胞凋亡变化及共聚焦显微镜观察线粒体膜电位（MMP）的改变。结；转染后48hhTERT．shRNA组hTERTmRNA相对表达量为0．32±0．03，hTERT蛋白表达的平均灰月值为209．604-17．10，端粒酶A450～A630值为2．242±0．284，较其他对照组均明显降低（P〈0．05P〈0．01）；G0／G1期细胞为（49．374-1．63）％，增殖指数为（50．60±1．57）％，凋亡指数为22．2％，莲其他对照组均明显增高（P〈0．05，P〈0．01）；凋亡细胞体积明显缩小，表面突起和微绒毛减少，甚!消失，线粒体Rhodamine123平均荧光强度为719．994-17．08，MMP水平显著下降（P〈0．01）。结tRNA干扰（RNAi）沉默hTERT基因能有效抑抑制SW480肿瘤细胞生长，并降低端粒酶活性，诱{肿瘤细胞凋亡。

Objective To observe the bionomics effects of short hairpin RNA （shRNA） fragment of human telomerase reverse transcriptase （hTERT） gene on human SW480 tumor cell line. Methods The human colorectal carcinoma cell line SW480 was transfected with recombinant eukaryotic expression plas- mid pGPU6/GFP/Neo-hTERT shRNA （hTERT-shRNA） by LipofectamineTM 2000, and cells were divided by four groups with 3 repetitive holes in each group. The hTERT mRNA and protein expression levels in SW480 cells were detected by reverse transcription-polymerase chain reaction （RT-PCR） and immunocyto- chemistry respectively, and the telomerase activity was detected by PCR-TRAP-ELISA. The effects of shRNA on cell cycle were analyzed by flow cytometry （FCM）. terminal-deoxynucleotidyl transferase media- ted nick end labeling （TUNEL） and transmission electron microscopy were used to observe the changes in SW480 cells apoptosis and mitochondrial membrane potential （MMP）. Results Forty-eight h after trans- feetion, hTERT mRNA relative expression was O. 32 ~ 0. 03, hTERT protein average gray scale was 209. 60 ~ 17. 10, A450-630 nm value of telomerase was 2. 242 ~ O. 284 in hTERT-shRNA group, which were reduced remarkably as compared with other three groups （P 〈 0. 05 ,P 〈 0. 01 ）. The cells number at phase G0/G1 was （49. 37 ~ 1.63 ） % , the proliferation index was （ 50. 60 ＋ 1.57 ） % , and the apoptotie index was 22. 2% in hTERT-shRNA group, which were increased obviously as compared with other three groups （P 〈 0. 05, P 〈 0. 01 ）. Under the transmission electron microscopy, apoptotic cells were found, the cellular volume got smaller, and the protrusion and minovillus were reduced, some even disappeared. The fluorescent intensity of Rhodamine 123 was 719. 99 ~ 17. 08, and MMP was reduced markedly in hTERT-shRNA group as compared with control groups （ P 〈 O. 01 ）. Conclusion RNA interference （ RNAi ） silencing hTERT gene can effectively inhibit SW480 cell growth, reduce the activity of telomerase and induce tumor cells anontosis.