利用腫瘤特異性和可熱調(diào)控的siRNA表達(dá)載體條件性敲除基因
發(fā)布時(shí)間：2018-11-09 13:05
【摘要】：RNAi是在生物進(jìn)化上高度保守的特異性轉(zhuǎn)錄后基因沉默過程,并已經(jīng)成為強(qiáng)大的基因操作工具。它幾乎可以用來(lái)研究任何一種基因的分子途徑以及表型和基因型之間的關(guān)系。但仍存在些許不足之處,嚴(yán)重限制了RNAi在基因治療領(lǐng)域中的廣泛應(yīng)用。其中RNAi在時(shí)間和空間上的不可控性以及非靶向性是需要亟待解決的難題。誘導(dǎo)型RNAi技術(shù)可以通過誘導(dǎo)物介導(dǎo)sh RNA的表達(dá)進(jìn)而實(shí)現(xiàn)對(duì)靶基因的有效調(diào)控。與誘導(dǎo)型調(diào)控系統(tǒng)結(jié)合的RNAi體系作用機(jī)制更精細(xì),這可能會(huì)開辟出一個(gè)更廣闊的應(yīng)用領(lǐng)域。本研究選取合適的熱休克元件和腫瘤特異性啟動(dòng)子,將兩者有效結(jié)合,構(gòu)建一種溫度誘導(dǎo)型的RNAi質(zhì)粒載體。具體實(shí)驗(yàn)內(nèi)容和結(jié)果如下:①選擇h TERT腫瘤特異性啟動(dòng)子的核心序列,它幾乎只在大多數(shù)的癌癥細(xì)胞中啟動(dòng)表達(dá)。熱休克蛋白啟動(dòng)子中含有可以響應(yīng)熱的熱休克元件,實(shí)驗(yàn)中選擇三種熱休克元件:一種來(lái)源于小HSP家族的Hsp27;另一種是三個(gè)“NGAAN-NTTC”HSE基本單位的重復(fù)排列;最后一種是由“AGAAC”和“GTTCT”交替排列得到的HSE序列。將HSE序列可以插入到h TERT啟動(dòng)子序列-65和-85位點(diǎn)之間或者放在首位。②構(gòu)建ph TERTp-EGFP、ph TERT/HSE(AB)p-EGFP、ph TERT/HSE(MN)p-EGFP和ph TERT/HSE(XY)p-EGFP四種質(zhì)粒,并將其轉(zhuǎn)染至MCF-7細(xì)胞中,通過檢測(cè)EGFP的蛋白表達(dá)量比較不同啟動(dòng)子的啟動(dòng)能力。數(shù)據(jù)顯示h TERT/HSE(MN)和h TERT/HSE(XY)啟動(dòng)子既可以降低h TERT啟動(dòng)子的本底表達(dá)水平又可以很好的響應(yīng)過高熱。此外,這兩種啟動(dòng)子在Ha Ca T和HL7702人正常細(xì)胞中表達(dá)EGFP的量遠(yuǎn)遠(yuǎn)低于Hsp70啟動(dòng)子,這表明所構(gòu)建的ph TERTp-EGFP和ph TERT/HSEp-EGFP質(zhì)粒具有良好的腫瘤特異性。③構(gòu)建p Mh TERTpsi SNCG和p Mh TERT/HSEpsi SNCG質(zhì)粒載體,并選擇MCF-7和Hep G2為實(shí)驗(yàn)細(xì)胞,以SNCG為靶基因,分別從RNA和蛋白水平上檢測(cè)這種表達(dá)系統(tǒng)的敲除效果。實(shí)驗(yàn)結(jié)果顯示無(wú)論是在MCF-7還是在Hep G2細(xì)胞中,p Mh TERT/HSE(MN)psi SNCG和p Mh TERT/HSE(XY)psi SNCG質(zhì)粒在37℃條件下幾乎不能敲除SNCG,而當(dāng)經(jīng)歷過熱誘導(dǎo),這兩種質(zhì)粒沉默SNCG基因的能力明顯提高,敲除率分別在50%和60%以上。
[Abstract]:RNAi is a highly conserved process of specific post-transcriptional gene silencing in biological evolution and has become a powerful tool for gene manipulation. It can be used to study the molecular pathways of almost any gene and the relationship between phenotypes and genotypes. However, there are still some deficiencies, which seriously limit the wide application of RNAi in gene therapy. The uncontrollability and non-targeting of RNAi in time and space are difficult problems to be solved. Inducible RNAi technique can effectively regulate the target gene by inducing the expression of sh RNA. The mechanism of RNAi system combined with inducible regulation system is more refined, which may open up a wider application field. In this study, the suitable heat shock elements and tumor specific promoters were selected and combined effectively to construct a temperature-induced RNAi plasmid vector. The specific experimental contents and results are as follows: 1 the core sequence of h TERT tumor specific promoter is selected, which only activates expression in most cancer cells. Heat shock protein promoters contain heat shock elements that can respond to heat. Three heat shock elements are selected in the experiment: one is Hsp27; from small HSP family and the other is repetitive arrangement of three basic units of "NGAAN-NTTC" HSE; The last is the HSE sequence, which is arranged alternately by "AGAAC" and "GTTCT". The HSE sequence can be inserted between the -65 and -85 sites of the h TERT promoter sequence or put in the first place. 2 to construct four kinds of plasmids of ph TERTp-EGFP,ph TERT/HSE (AB) p-EGFP, TERT/HSE (MN) p-EGFP and ph TERT/HSE (XY) p-EGFP. After transfection into MCF-7 cells, the promoter ability of different promoters was compared by detecting the protein expression of EGFP. The data showed that h TERT/HSE (MN) and h TERT/HSE (XY) promoters could not only reduce the background expression level of h TERT promoter but also respond to hyperthermia. In addition, the expression of EGFP in Ha Ca T and HL7702 human normal cells was much lower than that in Hsp70 promoter. This indicated that the constructed plasmids of ph TERTp-EGFP and ph TERT/HSEp-EGFP had good tumor specificity. 3 the plasmid vectors of p Mh TERTpsi SNCG and p Mh TERT/HSEpsi SNCG were constructed, and MCF-7 and Hep G2 were selected as experimental cells and SNCG as target gene. The knockout effects of the expression system were detected at RNA and protein levels, respectively. The results showed that both, p Mh TERT/HSE (MN) psi SNCG and p Mh TERT/HSE (XY) psi SNCG plasmids could hardly knock out SNCG, at 37 鈩，
本文編號(hào)：2320497
[Abstract]:RNAi is a highly conserved process of specific post-transcriptional gene silencing in biological evolution and has become a powerful tool for gene manipulation. It can be used to study the molecular pathways of almost any gene and the relationship between phenotypes and genotypes. However, there are still some deficiencies, which seriously limit the wide application of RNAi in gene therapy. The uncontrollability and non-targeting of RNAi in time and space are difficult problems to be solved. Inducible RNAi technique can effectively regulate the target gene by inducing the expression of sh RNA. The mechanism of RNAi system combined with inducible regulation system is more refined, which may open up a wider application field. In this study, the suitable heat shock elements and tumor specific promoters were selected and combined effectively to construct a temperature-induced RNAi plasmid vector. The specific experimental contents and results are as follows: 1 the core sequence of h TERT tumor specific promoter is selected, which only activates expression in most cancer cells. Heat shock protein promoters contain heat shock elements that can respond to heat. Three heat shock elements are selected in the experiment: one is Hsp27; from small HSP family and the other is repetitive arrangement of three basic units of "NGAAN-NTTC" HSE; The last is the HSE sequence, which is arranged alternately by "AGAAC" and "GTTCT". The HSE sequence can be inserted between the -65 and -85 sites of the h TERT promoter sequence or put in the first place. 2 to construct four kinds of plasmids of ph TERTp-EGFP,ph TERT/HSE (AB) p-EGFP, TERT/HSE (MN) p-EGFP and ph TERT/HSE (XY) p-EGFP. After transfection into MCF-7 cells, the promoter ability of different promoters was compared by detecting the protein expression of EGFP. The data showed that h TERT/HSE (MN) and h TERT/HSE (XY) promoters could not only reduce the background expression level of h TERT promoter but also respond to hyperthermia. In addition, the expression of EGFP in Ha Ca T and HL7702 human normal cells was much lower than that in Hsp70 promoter. This indicated that the constructed plasmids of ph TERTp-EGFP and ph TERT/HSEp-EGFP had good tumor specificity. 3 the plasmid vectors of p Mh TERTpsi SNCG and p Mh TERT/HSEpsi SNCG were constructed, and MCF-7 and Hep G2 were selected as experimental cells and SNCG as target gene. The knockout effects of the expression system were detected at RNA and protein levels, respectively. The results showed that both, p Mh TERT/HSE (MN) psi SNCG and p Mh TERT/HSE (XY) psi SNCG plasmids could hardly knock out SNCG, at 37 鈩，
本文編號(hào)：2320497
本文鏈接：http://sikaile.net/huliyixuelunwen/2320497.html
最近更新
教材專著
