靶向人表皮生长因子受体RNAi表达载体的构建与鉴定

发布时间：2018-05-12 21:29

  本文选题：hEGFR + hTERT　； 参考：《郑州大学》2010年硕士论文

【摘要】： RNA干扰(RNA interference, RNAi)是指在转录后水平有效沉默特异性基因的表达,是生物遗传控制网络中的重要通路之一,已被广泛用于肿瘤的治疗研究。人表皮生长因子受体(human epidermal growth factor receptor, hEGFR)在多种肿瘤细胞中过度表达,参与肿瘤形成发展的多个过程,是治疗肿瘤的重要靶点之一。本研究鉴于RNAi的多项优势以及hEGFR作为肿瘤靶点的潜在应用价值,以RNAi作为降低hEGFR基因表达的途径,探讨其对人类肝癌细胞SMMC-7721的生长抑制作用。 靶向hEGFR的单基因沉默载体为pRS-EGFR系列载体,阴性对照为Rs1。我们将以上载体瞬时转染至人类肝癌细胞SMMC-7721,通过RT-PCR实验研究了hEGFR基因的表达情况；同时我们对转染单基因沉默载体的肝癌细胞进行了稳定筛选,2周左右得到稳定表达细胞株,对其进行了RT-PCR和MTT实验。结果表明,与阴性对照组相比,瞬时转染和稳定表达重组质粒pRS-EGFR-sh的SMMC-7721肝癌细胞中hEGFR的mRNA相对表达量分别下降了48%、46%、13%、5%和51%、44%、21%、10%；MTT实验结果显示：稳定表达RNAi载体的肝癌细胞生长速度受到不同程度的抑制。这提示我们,RNAi的影响因素是多方面而且未知的,随着分子生物各项技术和基因组学的发展,RNAi有待于更深入的研究和完善。 肿肿瘤的发病涉及到多个信号通路,它们相互联系、相互作用,组成一个分子网络体系,因此针对单个靶点进行抗肿瘤治疗很有可能收效甚微。肿瘤的多靶点治疗可以从多方面阻滞癌细胞增殖,符合肿瘤的发病规律和治病要求,是抗肿瘤治疗的发展趋势。hTERT (human telomerase reverse transcriptase, hTERT)作为端粒酶活性的限制组分,在多种肿瘤细胞中表达显著增强,是抗肿瘤治疗的另一理想靶点。有文献报道,EGFR过度表达可以激活端粒酶活性,阻断EGFR信号通路后,会伴随有端粒酶活性的下降以及TERT表达减弱。本研究根据EGFR与TERT之间的密切联系,以及肿瘤多靶点治疗的重要意义,将靶向hEGFR和hTERT的shRNA构建至同一pRS载体,即RNA干扰双基因表达载体,以达到多方位、低耐药、高力度抗肿瘤的目的。 以hTERT为作用靶点的RNA干扰载体分别命名为为shRNA-T系列载体。用EcoRⅠ和HindⅢ双酶切pRS-EGFR-sh载体,回收含有hEGFR-shRNA序列的小片段,并将其小片段插入shRNA-T系列载体的EcoRⅠ位点,构建同时表达hTERT-shRNA和hEGFR-shRNA的双基因RNA干扰载体。电泳图谱结果呈现预期的DNA条带,说明双基因RNA干扰载体构建成功。
[Abstract]:RNA interference is one of the important pathways in the biological genetic control network and has been widely used in the treatment of cancer. It refers to the effective silencing of specific gene expression at the post-transcriptional level and is one of the important pathways in the biological genetic control network. Human epidermal growth factor receptor (epidermal growth factor receptor, hEGFR) is overexpressed in various tumor cells, which is involved in many processes of tumor formation and development, and is one of the important targets in the treatment of tumor. In view of the multiple advantages of RNAi and the potential application value of hEGFR as a tumor target, RNAi was used as a way to reduce the expression of hEGFR gene to investigate the inhibitory effect of RNAi on the growth of human hepatoma cell line SMMC-7721. The single gene silencing vector targeting hEGFR was pRS-EGFR series vector, while the negative control was Rs1. We transiently transfected SMMC-7721 into human hepatoma cell line SMMC-7721 and studied the expression of hEGFR gene by RT-PCR assay. At the same time, we stably screened the hepatoma cells transfected with single gene silencing vector for about 2 weeks to obtain stable expression cell lines. RT-PCR and MTT experiments were carried out. The results showed that, compared with the negative control group, The relative expression of hEGFR mRNA in SMMC-7721 hepatoma cells with transient transfection and stable expression of recombinant plasmid pRS-EGFR-sh decreased by 48% and 46%, respectively. The results showed that the growth rate of hepatoma cells with stable expression of RNAi was inhibited in varying degrees. It is suggested that the influencing factors of RNAi are various and unknown. With the development of molecular biology and genomics, RNAi needs to be further studied and improved. Multiple signal pathways are involved in the pathogenesis of tumor, which interact with each other and form a molecular network system. Therefore, anti-tumor therapy for a single target is likely to have little effect. The multitarget therapy of tumor can block the proliferation of cancer cells from many aspects, which accords with the rule of tumor development and the requirement of treatment. It is the development trend of anti-tumor therapy, such as hTERT telomerase reverse transcriptase, hTERT) as the limiting component of telomerase activity. The increased expression in various tumor cells is another ideal target for anti-tumor therapy. It has been reported that overexpression of EGFR can activate telomerase activity. After blocking EGFR signaling pathway, telomerase activity decreases and TERT expression weakens. In this study, according to the close relationship between EGFR and TERT, and the significance of multi-target tumor therapy, the shRNA targeting hEGFR and hTERT was constructed into the same pRS vector, that is, RNA interference double gene expression vector, in order to achieve multidirectional and low drug resistance. The aim of high-intensity anti-tumor. RNA interference vectors with hTERT as target were named as shRNA-T series carriers respectively. The pRS-EGFR-sh vector was digested with EcoR 鈪，

本文编号：1880227