摘要
目的研究端粒酶逆转录酶(hTERT)RNA干扰对结肠癌HCT116细胞生物学行为的影响。方法将HCT116细胞分为转染组(转染重组质粒真核表达载体)、对照组(转染空载体质粒)和未转染组。PCR方法检测hTERT干扰序列,RT-PCR方法检测hTERT表达,HE染色、生长曲线和流式细胞术方法分别检测细胞形态、细胞增殖和细胞周期,β-半乳糖苷酶染色方法检测细胞衰老,AnnexinⅤ/PI染色流式细胞光度术检测细胞凋亡。结果转染细胞内均存在hTERT干扰序列,hTERT干扰率为21.5%;与未转染细胞相比,转染细胞核质比明显缩小,增殖率显著下降(P<0.05),衰老细胞和G2-M期细胞明显增加(P<0.05)。hTERT干扰显著增加结肠癌细胞凋亡(P<0.05),而对照细胞各指标均无显著变化。结论hTERT干扰显著影响结肠癌细胞的生物学行为。
Objective To study the effect of human telomerase reverse transcriptase （hTERT） RNA interference （RNAi） on biological characteristics of colon carcinoma cell line HCT 116. Methods Small hairpin hTERT （shTERT） sequence was identified by polymerase chain reaction （PCR） method; hTERT expression, morphological features, cell proliferation and replicative senescence were determined by reverse transcription PCR （RT-PCR） , hematoxylin-eosin （HE） straining, cell growth curve and β-galactosidase （β-Gal） staining, respectively. Cell cycle and apoptosis were respectively identified by flow eytometry after proidium iodide （PI） straining and Annexin V/PI double straining. Results shRNA were all found in 2 cell clones transformed by the recombined plasmid pSileneer 3. 1-H1 neo-shTERT. The interference rate of hTERT was 21.5%. The cell in G2-M phase and percentage of replieative senscence cell significantly increased. The nucleus/cytoplasm ratio of the cell was obviously decreased after hTERT RNAi treatment. Moreover, apoptosis of colon carcinoma cell was string increased by hTERT RNAi （P 〈 0.05）. However, there was no remarkably change between control cell and untransformed cell. Conclusion hTERT RNAi has significant effects on biological characteristics of colon carcinoma cell line HCT 116.
出处
《胃肠病学和肝病学杂志》
CAS
2009年第3期243-246,共4页
Chinese Journal of Gastroenterology and Hepatology
