利用siRNA真核表達(dá)載體干擾MGMT基因表達(dá)的初步研究
發(fā)布時(shí)間：2019-02-12 10:52
【摘要】： 烷化劑作用于細(xì)胞DNA,產(chǎn)生O~6-烷基鳥(niǎo)嘌呤,引起DNA損傷,殺傷細(xì)胞。而腫瘤細(xì)胞內(nèi)的O~6-甲基鳥(niǎo)嘌呤-DNA甲基轉(zhuǎn)移酶(MGMT)能夠參與修復(fù)腫瘤細(xì)胞的DNA烷化損傷,使腫瘤細(xì)胞對(duì)亞硝脲類烷化劑產(chǎn)生耐藥性,是降低烷化劑藥物對(duì)腫瘤細(xì)胞殺滅效應(yīng)的關(guān)鍵因素之一,這是許多腫瘤細(xì)胞對(duì)亞硝脲類烷化劑耐藥的分子基礎(chǔ)。 為了提高腫瘤細(xì)胞對(duì)烷化劑藥物的敏感性,Kreklau等使用抑制劑抑制腫瘤細(xì)胞中MGMT的活性。但是使用抑制劑后,外周血單核細(xì)胞MGMT的活性受到抑制。其抑制作用是免疫反應(yīng)性,長(zhǎng)期的蓄積會(huì)導(dǎo)致骨髓毒性。Yoshinaga等曾報(bào)道用反義核酸技術(shù)、核酶技術(shù)抑制MGMT基因的表達(dá),可以逆轉(zhuǎn)腫瘤細(xì)胞對(duì)烷化劑的耐藥性,但效應(yīng)較低,選擇序列比較盲目。近年來(lái),RNA干擾成為最有效的基因沉默方式,siRNA具有特殊的雙鏈互補(bǔ)RNA結(jié)構(gòu),尤其siRNA真核表達(dá)載體,可以在細(xì)胞內(nèi)較長(zhǎng)時(shí)間發(fā)揮作用,比反義RNA穩(wěn)定,siRNA較之反義RNA技術(shù)及核酶具有獨(dú)特的、可能更理想的基因表達(dá)調(diào)控效果,應(yīng)用前景廣闊。 為了比較不同靶序列的RNAi作用,本研究首先合成3條編碼發(fā)夾siRNA序列的單鏈DNA,克隆到pRNATin-H1.2/Neo載體H1.2啟動(dòng)子的下游,構(gòu)建成含目的基因片段的重組質(zhì)粒pRNATin- H1.2/NeoMGMT siRNA,以脂質(zhì)體Lipofectimine 2000為介質(zhì)轉(zhuǎn)染人宮頸癌HeLa S3細(xì)胞,利用半定量RT-PCR,實(shí)時(shí)定量RT-PCR檢測(cè)MGMT基因的抑制作用,采用MTT法,測(cè)定siRNA真核表達(dá)載體轉(zhuǎn)染前后HeLa S3對(duì)烷化劑BCNU的敏感性變化,從而為利用RNA干擾提高腫瘤細(xì)胞對(duì)烷化劑的敏感性提供初步的實(shí)驗(yàn)證據(jù)。主要結(jié)果如下: 1、成功構(gòu)建siRNA真核表達(dá)載體pRNATin-H1.2/NeoMGMT siRNA,重組質(zhì)粒轉(zhuǎn)化后得到陽(yáng)性克隆,抽提質(zhì)粒,經(jīng)PCR擴(kuò)增,在2%瓊脂糖凝膠電泳結(jié)果顯示:PCR產(chǎn)物大小210bp,產(chǎn)物大小與設(shè)計(jì)的完全相同。測(cè)序結(jié)果證明序列正確。 2、轉(zhuǎn)染后HeLa S3細(xì)胞的GFP表達(dá):在倒置熒光顯微鏡下觀察,經(jīng)LipofectimineTM2000轉(zhuǎn)染的HeLa S3細(xì)胞在轉(zhuǎn)染后48h,在熒光顯微鏡下可見(jiàn)細(xì)胞呈現(xiàn)
[Abstract]:Alkylating agent acts on cell DNA, to produce OF6-alkyl guanine, causing DNA damage and killing cells. However, OF6-methylguanine DNA methyltransferase (MGMT) can be involved in the repair of DNA alkylation damage in tumor cells and make tumor cells resistant to nitrite ureas alkylators. It is one of the key factors to reduce the killing effect of alkylating agents on tumor cells, which is the molecular basis of resistance of many tumor cells to nitrite alkylates. In order to improve the sensitivity of tumor cells to alkylating agents, Kreklau and other inhibitors inhibit the activity of MGMT in tumor cells. However, the activity of MGMT in peripheral blood monocytes was inhibited after the use of inhibitors. Antisense nucleic acid and ribozyme techniques have been reported to inhibit the expression of MGMT gene, which can reverse the resistance of tumor cells to alkylating agents, but the effect is low. The selection sequence is blind. In recent years, RNA interference has become the most effective way of gene silencing. SiRNA has a special double-stranded complementary RNA structure, especially siRNA eukaryotic expression vector, which can play a role in cells for a long time and is more stable than antisense RNA. Compared with antisense RNA technique and ribozyme, siRNA has a unique effect on gene expression regulation. In order to compare the RNAi effects of different target sequences, three single-stranded DNA, encoding hairpin siRNA sequences were synthesized and cloned into the downstream of the pRNATin-H1.2/Neo vector H1.2 promoter. The recombinant plasmid pRNATin- H1.2/NeoMGMT siRNA, containing the target gene fragment was constructed and transfected into human cervical cancer HeLa S3 cells using liposome Lipofectimine 2000 as the medium. The inhibition of MGMT gene was detected by semi-quantitative RT-PCR, real-time quantitative RT-PCR, and MTT assay was used. The sensitivity of HeLa S3 to alkylating agent BCNU was determined before and after transfection of siRNA eukaryotic expression vector, thus providing preliminary experimental evidence for enhancing the sensitivity of tumor cells to alkylating agent by RNA interference. The main results are as follows: 1. The siRNA eukaryotic expression vector pRNATin-H1.2/NeoMGMT siRNA, recombinant plasmid was successfully constructed and transformed into a positive clone. The plasmid was extracted and amplified by PCR. The results of 2% agarose gel electrophoresis showed that the product size of PCR was 210bpand the size of the product was the same as that designed. The sequencing results show that the sequence is correct. 2. GFP expression of HeLa S3 cells after transfection: observed under inverted fluorescence microscope, the cells of HeLa S3 transfected with LipofectimineTM2000 were observed under fluorescence microscope at 48h after transfection.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346
本文編號(hào)：2420374
[Abstract]:Alkylating agent acts on cell DNA, to produce OF6-alkyl guanine, causing DNA damage and killing cells. However, OF6-methylguanine DNA methyltransferase (MGMT) can be involved in the repair of DNA alkylation damage in tumor cells and make tumor cells resistant to nitrite ureas alkylators. It is one of the key factors to reduce the killing effect of alkylating agents on tumor cells, which is the molecular basis of resistance of many tumor cells to nitrite alkylates. In order to improve the sensitivity of tumor cells to alkylating agents, Kreklau and other inhibitors inhibit the activity of MGMT in tumor cells. However, the activity of MGMT in peripheral blood monocytes was inhibited after the use of inhibitors. Antisense nucleic acid and ribozyme techniques have been reported to inhibit the expression of MGMT gene, which can reverse the resistance of tumor cells to alkylating agents, but the effect is low. The selection sequence is blind. In recent years, RNA interference has become the most effective way of gene silencing. SiRNA has a special double-stranded complementary RNA structure, especially siRNA eukaryotic expression vector, which can play a role in cells for a long time and is more stable than antisense RNA. Compared with antisense RNA technique and ribozyme, siRNA has a unique effect on gene expression regulation. In order to compare the RNAi effects of different target sequences, three single-stranded DNA, encoding hairpin siRNA sequences were synthesized and cloned into the downstream of the pRNATin-H1.2/Neo vector H1.2 promoter. The recombinant plasmid pRNATin- H1.2/NeoMGMT siRNA, containing the target gene fragment was constructed and transfected into human cervical cancer HeLa S3 cells using liposome Lipofectimine 2000 as the medium. The inhibition of MGMT gene was detected by semi-quantitative RT-PCR, real-time quantitative RT-PCR, and MTT assay was used. The sensitivity of HeLa S3 to alkylating agent BCNU was determined before and after transfection of siRNA eukaryotic expression vector, thus providing preliminary experimental evidence for enhancing the sensitivity of tumor cells to alkylating agent by RNA interference. The main results are as follows: 1. The siRNA eukaryotic expression vector pRNATin-H1.2/NeoMGMT siRNA, recombinant plasmid was successfully constructed and transformed into a positive clone. The plasmid was extracted and amplified by PCR. The results of 2% agarose gel electrophoresis showed that the product size of PCR was 210bpand the size of the product was the same as that designed. The sequencing results show that the sequence is correct. 2. GFP expression of HeLa S3 cells after transfection: observed under inverted fluorescence microscope, the cells of HeLa S3 transfected with LipofectimineTM2000 were observed under fluorescence microscope at 48h after transfection.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346
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