發(fā)布時間：2018-05-12 21:29

  本文選題：hEGFR + hTERT��； 參考：《鄭州大學》2010年碩士論文

【摘要】： RNA干擾(RNA interference, RNAi)是指在轉(zhuǎn)錄后水平有效沉默特異性基因的表達,是生物遺傳控制網(wǎng)絡(luò)中的重要通路之一,已被廣泛用于腫瘤的治療研究。人表皮生長因子受體(human epidermal growth factor receptor, hEGFR)在多種腫瘤細胞中過度表達,參與腫瘤形成發(fā)展的多個過程,是治療腫瘤的重要靶點之一。本研究鑒于RNAi的多項優(yōu)勢以及hEGFR作為腫瘤靶點的潛在應用價值,以RNAi作為降低hEGFR基因表達的途徑,探討其對人類肝癌細胞SMMC-7721的生長抑制作用。 靶向hEGFR的單基因沉默載體為pRS-EGFR系列載體,陰性對照為Rs1。我們將以上載體瞬時轉(zhuǎn)染至人類肝癌細胞SMMC-7721,通過RT-PCR實驗研究了hEGFR基因的表達情況；同時我們對轉(zhuǎn)染單基因沉默載體的肝癌細胞進行了穩(wěn)定篩選,2周左右得到穩(wěn)定表達細胞株,對其進行了RT-PCR和MTT實驗。結(jié)果表明,與陰性對照組相比,瞬時轉(zhuǎn)染和穩(wěn)定表達重組質(zhì)粒pRS-EGFR-sh的SMMC-7721肝癌細胞中hEGFR的mRNA相對表達量分別下降了48%、46%、13%、5%和51%、44%、21%、10%；MTT實驗結(jié)果顯示：穩(wěn)定表達RNAi載體的肝癌細胞生長速度受到不同程度的抑制。這提示我們,RNAi的影響因素是多方面而且未知的,隨著分子生物各項技術(shù)和基因組學的發(fā)展,RNAi有待于更深入的研究和完善。 腫腫瘤的發(fā)病涉及到多個信號通路,它們相互聯(lián)系、相互作用,組成一個分子網(wǎng)絡(luò)體系,因此針對單個靶點進行抗腫瘤治療很有可能收效甚微。腫瘤的多靶點治療可以從多方面阻滯癌細胞增殖,符合腫瘤的發(fā)病規(guī)律和治病要求,是抗腫瘤治療的發(fā)展趨勢。hTERT (human telomerase reverse transcriptase, hTERT)作為端粒酶活性的限制組分,在多種腫瘤細胞中表達顯著增強,是抗腫瘤治療的另一理想靶點。有文獻報道,EGFR過度表達可以激活端粒酶活性,阻斷EGFR信號通路后,會伴隨有端粒酶活性的下降以及TERT表達減弱。本研究根據(jù)EGFR與TERT之間的密切聯(lián)系,以及腫瘤多靶點治療的重要意義,將靶向hEGFR和hTERT的shRNA構(gòu)建至同一pRS載體,即RNA干擾雙基因表達載體,以達到多方位、低耐藥、高力度抗腫瘤的目的。 以hTERT為作用靶點的RNA干擾載體分別命名為為shRNA-T系列載體。用EcoRⅠ和HindⅢ雙酶切pRS-EGFR-sh載體,回收含有hEGFR-shRNA序列的小片段,并將其小片段插入shRNA-T系列載體的EcoRⅠ位點,構(gòu)建同時表達hTERT-shRNA和hEGFR-shRNA的雙基因RNA干擾載體。電泳圖譜結(jié)果呈現(xiàn)預期的DNA條帶,說明雙基因RNA干擾載體構(gòu)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，

本文編號：1880227