利用siRNA真核表达载体干扰MGMT基因表达的初步研究
发布时间：2019-02-12 10:52
【摘要】： 烷化剂作用于细胞DNA,产生O~6-烷基鸟嘌呤,引起DNA损伤,杀伤细胞。而肿瘤细胞内的O~6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)能够参与修复肿瘤细胞的DNA烷化损伤,使肿瘤细胞对亚硝脲类烷化剂产生耐药性,是降低烷化剂药物对肿瘤细胞杀灭效应的关键因素之一,这是许多肿瘤细胞对亚硝脲类烷化剂耐药的分子基础。 为了提高肿瘤细胞对烷化剂药物的敏感性,Kreklau等使用抑制剂抑制肿瘤细胞中MGMT的活性。但是使用抑制剂后,外周血单核细胞MGMT的活性受到抑制。其抑制作用是免疫反应性,长期的蓄积会导致骨髓毒性。Yoshinaga等曾报道用反义核酸技术、核酶技术抑制MGMT基因的表达,可以逆转肿瘤细胞对烷化剂的耐药性,但效应较低,选择序列比较盲目。近年来,RNA干扰成为最有效的基因沉默方式,siRNA具有特殊的双链互补RNA结构,尤其siRNA真核表达载体,可以在细胞内较长时间发挥作用,比反义RNA稳定,siRNA较之反义RNA技术及核酶具有独特的、可能更理想的基因表达调控效果,应用前景广阔。 为了比较不同靶序列的RNAi作用,本研究首先合成3条编码发夹siRNA序列的单链DNA,克隆到pRNATin-H1.2/Neo载体H1.2启动子的下游,构建成含目的基因片段的重组质粒pRNATin- H1.2/NeoMGMT siRNA,以脂质体Lipofectimine 2000为介质转染人宫颈癌HeLa S3细胞,利用半定量RT-PCR,实时定量RT-PCR检测MGMT基因的抑制作用,采用MTT法,测定siRNA真核表达载体转染前后HeLa S3对烷化剂BCNU的敏感性变化,从而为利用RNA干扰提高肿瘤细胞对烷化剂的敏感性提供初步的实验证据。主要结果如下: 1、成功构建siRNA真核表达载体pRNATin-H1.2/NeoMGMT siRNA,重组质粒转化后得到阳性克隆,抽提质粒,经PCR扩增,在2%琼脂糖凝胶电泳结果显示:PCR产物大小210bp,产物大小与设计的完全相同。测序结果证明序列正确。 2、转染后HeLa S3细胞的GFP表达:在倒置荧光显微镜下观察,经LipofectimineTM2000转染的HeLa S3细胞在转染后48h,在荧光显微镜下可见细胞呈现
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346
本文编号：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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346
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