ILKmRNA在结肠癌细胞株中表达及其稳定表达RNA干扰载体构建.docVIP

ILKmRNA在结肠癌细胞株中表达及其稳定表达RNA干扰载体构建 　【摘要】 　　［目的］ 检测结肠癌细胞株中ILK的mRNA水平，并构建针对ILK基因的特异性RNA干扰(RNAi)表达载体。［方法］ 通过RT-PCR检测ILK基因在三种结肠癌细胞株（SW480、LO-VO及HT29）mRNA的表达；根据GenBank提供的ILK基因mRNA序列，设计具有小发夹结构的两条DNA序列，化学合成后经退火形成双链DNA片段，连接到经HindⅢ／BglⅡ酶切线性化的pSUPER-neo-EGFP／H1（pSNE/H1）质粒上，形成重组载体pSNE-ILK，转化大肠杆菌DH5α，提取质粒经HindⅢ/EcoRⅠ双酶切鉴定和测序鉴定，稳定转染结肠癌细胞HT29。［结果］ ①RT-PCR检测，ILK基因在三种结肠癌细胞株中均有表达，ILK/β-actin电泳条带强度比：SW480为0.63，HT29为0.65，LO-VO为0.33。②成功构建了针对ILK基因的特异性RNA干扰载体pSNE-ILK和对照的无关基因载体pSNE-Control，并稳定转染结肠癌细胞株HT-29。［结论］ 成功构建了针对ILK基因的特异性RNAi载体，并稳定转染HT29细胞，能有效抑制ILK基因，为下一步探讨ILK基因在结肠癌进程中的作用和结肠癌的基因治疗奠定了基础。 【关键词】 RNA干扰　载体构建　RT-PCR　整合素连接激酶 　　Expression of ILK mRNA in Colon Carcinoma Cell Lines and Construction of ILK-RNAi Stable Expressing Vector Abstract： ［Purpose］ To investigate the expression of ILK mRNA in the colon carcinoma cell line and to construct novel ILK-RNAi(RNA interference) vector. ［Methods］ The expression of ILK mRNA was detected by RT-PCR in three colon carcinoma cell lines（SW480, LO-VO and HT29）. Specific short chain oligonucleotide was designed using the RNAi software according to the mRNA sequence provided by GeneBank，the oligonucleotides was gained through annealing after chemosynthesis and was inserted into HindⅢ/BglⅡlinearized pSUPER-neo-EGFP/H1（pSNE/H1）plasmids. The recombinant expression vector was evaluated using enzyme cutting and sequencing．The right vectors were stably transfected into HT29 cells． ［Results］ The numerical value of ILK/β-actin were detected to evaluate expression of ILK mRNA in the three colon carcinoma cell lines: SW480, 0.63; HT29, 0.65 and LO-VO, 0.33. pSNE-ILK and pSNE-Control had been constructed successfully.［Conclusion］ The ILK-targeted RNAi vector is constructed successfully then transfected into HT29 cells，and it can effectively inhibit the expression of target gene and may be potentially useful in gene therapy of ILK related colon carcinoma. Key words：RNA interference；vector construction；RT-PCR；integrin-linked kinase 免疫染色强度与肿瘤细胞的浸润深度、淋巴转移以及肿瘤的分级、分期呈正