结肠癌细胞中ap-2α抑制htert表达的实验研究-experimental study on ap - 2α inhibiting htert expression in colon cancer cells.docx

山西医科大学硕士学位论文 山西医科大学硕士学位论文 PAGE PAGE VI 细胞增殖情况； 5、TRAP-PCR 银染法检测 SW480 中转染 pcDNA3.1(+)-AP-2α 前后端粒酶活性。 结果： 1、酶切鉴定法及 DNA 结构测序均证明 pcDNA3.1(+)-Ap-2α 真核表达质粒为所 需要的目的质粒。 2、MTT 结果提示转染 Ap-2α-siRNA 后，HCT116 细胞增殖加快，48、72、96H 的增殖率分别为 27.1%、36.4%、42.1% 3、转染 pcDNA3.1(+)-AP-2α 至 SW480 细胞后，Ap-2α mRNA 及蛋白水平均显 著升高；hTERT mRNA 及蛋白水平均均明显下降，与转染前相比分别下降了 40.14%及 36.52%。； 4、转染 GV102-AP-2α-RNAi 至 HCT116 后，Ap-2α mRNA 含量降低；hTERT mRNA 含量升高，与转染前相比升高了 1.92 倍。 5、银染法示 SW480 转染 Ap-2α 基因后特征性条带变得不明显，端粒酶活性降 低。 6、转染 pcDNA3.1(+)-AP-2α 至 SW480 细胞后，Sp1 蛋白表达水平与之前相比 降低了 34%；转染 GV102-AP-2α-RNAi 至 HCT116 后，Sp1 蛋白表达水平升高 了 1.24 倍。 结论： AP-2α 对结肠癌细胞 HCT116 的增殖起到了抑制作用，具体的作用机制可 能是通过减少 SP-1 表达进而降低 hTERT 的表达水平，从而发挥抑制结肠癌细 胞增殖的作用。AP-2α 作为一种抑癌基因在本实验中也得到证实，为下一步结 肠癌的基因治疗提供了实验室理论依据。 关键词： 转录因子 Ap-2α、hTERT、SP1、结肠肿瘤、基因表达 Experimental study of AP-2α inhibit the expression of hTERT in colon cancer cells Abstract: Colon cancer , one of the higher mortality rate cancer in the world, has a very bad influence on human health and life. According to statistics, colon cancer mortality is in the second place in the Western developed countries and ranks fifth in our country. A multistage interference, multiple genes involved in the pathological process of colon cancer. AP-2α is a member of the transcription factor activated protein AP-2 family, which involved in a variety of tumor formation, migration and metastasis as a tumor suppressor gene. Telomerase catalytic subunit that is transcriptase (hTERT) highly expressed in tumor cells as the core of the telomerase, but lowly expressed in normal cells. Telomerase activation is the specificity of tumor formation ID. Therefore, in the field of intestinal tumors molecules, searching for a new prevention and treatment has become an important topic. Objective: 1．Observe the effection of AP-2α expression on proliferation in HCT116 cells. 2．Studies the transcription factor Ap-2α regulation of hTERT in colon cancer cell. 3．Research the telomerase activity after Ap-2α transfected in SW480 4．prelim