RNA干扰对WT1基因表达的抑制作用
发布时间：2019-02-15 10:09
【摘要】：目的研究siRNA(small interfering RNA,siRNA)对WT1基因的抑制作用,构建WT1基因siRNA载体,为进一步探讨WT1在白血病中的生物学功能及白血病的基因治疗奠定基础。 方法设计制备多对针对WT1基因的小干扰RNA(small interfering RNA, siRNA),通过阳离子脂质体Lipofectamine 2000转染乳腺癌细胞株MCF-7细胞,流式细胞仪检测转染效率,采用实时定量PCR(Real-time Quantitative Polymerase Chain Reaction, RQ-PCR)法测定WT1及内参β-actin mRNA的表达,筛选出最有效的siRNA,Western Blot测定WT1蛋白的表达。用不同浓度的最有效的WT1siRNA转染MCF-7细胞,观察不同浓度siRNA对RNAi效应的影响。用10nmol/L浓度的最有效siRNA转染MCF-7细胞后,继续培养24、48、72、96和120h收集细胞进行检测分析,观察RNAi效应的持续时间。并用流式细胞仪(FCM)检测WT1被干扰后,MCF-7细胞对长春新碱诱导凋亡的敏感性。根据最有效的siRNA序列,设计合成两条shRNA(small hair RNA, shRNA)的DNA模板单链,同时模板链两端分别设计不同的两个限制酶切位点。退火形成siRNA载体插入片断。用限制性内切酶将siRNA空载体线性化,T4连接酶将插入片断插入siRNA空质粒中。经酶切、PCR和测序的方法鉴定质粒是否成功。通过脂质体将装有WT1的干扰质粒和阴性对照质粒转入K562细胞中,以绿色荧光蛋白(GFP)基因为报告基因,用流式细胞仪和荧光显微镜观察转染效率和G418筛选4周后质粒表达效率。RT-PCR检测WT1基因表达变化,采用台盼兰拒染法、MTT比色法、甲基纤维素集落形成实验检测WT1基因干扰后对K562细胞生长的影响。通过流式细胞仪测定AnnexinV结合力观察WT1基因干扰后对K562细胞诱导凋亡的作用。 结果①脂质体转染MCF-7细胞的效率均值为98.1%(97.2～98.7%),所设计的8对siRNA中,4对能抑制WT1基因的表达,抑制效率在17.3%~89.79%之间。②5、10、50、100、150和200nmol/L浓度的siRNA转染MCF-7细胞后,干扰效率分别为91.00%、91.98%、81.52%、73.02%、77.53%、42.97%;在10nmol/L浓度siRNA作用下,可维持RNAi效应至少4天时间。③流式细胞仪测定Annexin V结合力的方法观察WT1基因被干扰后对长春新碱的诱导凋亡作用的敏感性,对
[Abstract]:Objective to study the inhibitory effect of siRNA (small interfering RNA,siRNA on WT1 gene and construct siRNA vector of WT1 gene for further study of the biological function of WT1 in leukemia and gene therapy for leukemia. Methods multiple pairs of small interfering RNA (small interfering RNA, siRNA), targeting WT1 gene were prepared and transfected into breast cancer cell line MCF-7 by cationic liposome Lipofectamine 2000. The transfection efficiency was detected by flow cytometry. Real time quantitative PCR (Real-time Quantitative Polymerase Chain Reaction, RQ-PCR) was used to detect the expression of WT1 and 尾-actin mRNA, and the most effective siRNA,Western Blot was selected to detect the expression of WT1 protein. MCF-7 cells were transfected with the most effective WT1siRNA at different concentrations to observe the effect of siRNA at different concentrations on the RNAi effect. After transfection of MCF-7 cells with the most effective siRNA at the concentration of 10nmol/L, the cells were collected and analyzed for the duration of RNAi effect. The sensitivity of MCF-7 cells to vincristine induced apoptosis after WT1 interference was detected by flow cytometry (FCM). According to the most effective siRNA sequence, two shRNA (small hair RNA, shRNA) single strands of DNA template were designed and synthesized. At the same time, two restriction endonuclease sites were designed at the two ends of the template chain. The siRNA carrier insert fragment is formed by annealing. The siRNA empty vector was linearized by restriction endonuclease, and the T 4 ligase was inserted into the empty siRNA plasmid. The plasmid was identified by enzyme digestion, PCR and sequencing. The interference plasmid containing WT1 and the negative control plasmid were transferred into K562 cells by liposome. The green fluorescent protein (GFP) gene was used as the reporter gene. Flow cytometry and fluorescence microscopy were used to observe the transfection efficiency and the efficiency of plasmid expression after G418 screening for 4 weeks. RT-PCR was used to detect the expression of WT1 gene. Trypan blue exclusion assay and MTT colorimetry were used to detect the expression of WT1 gene. The effect of WT1 gene interference on the growth of K562 cells was detected by colony forming assay of methylcellulose. The effect of WT1 gene interference on apoptosis of K562 cells was observed by flow cytometry (FCM). Results 1 the average efficiency of liposome transfection into MCF-7 cells was 98.1% (97.2n 98.7%). Of the 8 pairs of siRNA designed, 4 pairs could inhibit the expression of WT1 gene. The inhibitory efficiency was between 17.3% and 89.79%. The interference efficiency of siRNA transfected with MCF-7 cells was 91.00 and 91.980.The interference efficiency was 73.02and 77.537.530.The interference efficiency was 73.02and 42.97, respectively. The RNAi effect could be maintained by 10nmol/L concentration siRNA for at least 4 days. 3 the sensitivity of WT1 gene to the apoptosis induced by vincristine was observed by flow cytometry.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346
本文编号：2423228
[Abstract]:Objective to study the inhibitory effect of siRNA (small interfering RNA,siRNA on WT1 gene and construct siRNA vector of WT1 gene for further study of the biological function of WT1 in leukemia and gene therapy for leukemia. Methods multiple pairs of small interfering RNA (small interfering RNA, siRNA), targeting WT1 gene were prepared and transfected into breast cancer cell line MCF-7 by cationic liposome Lipofectamine 2000. The transfection efficiency was detected by flow cytometry. Real time quantitative PCR (Real-time Quantitative Polymerase Chain Reaction, RQ-PCR) was used to detect the expression of WT1 and 尾-actin mRNA, and the most effective siRNA,Western Blot was selected to detect the expression of WT1 protein. MCF-7 cells were transfected with the most effective WT1siRNA at different concentrations to observe the effect of siRNA at different concentrations on the RNAi effect. After transfection of MCF-7 cells with the most effective siRNA at the concentration of 10nmol/L, the cells were collected and analyzed for the duration of RNAi effect. The sensitivity of MCF-7 cells to vincristine induced apoptosis after WT1 interference was detected by flow cytometry (FCM). According to the most effective siRNA sequence, two shRNA (small hair RNA, shRNA) single strands of DNA template were designed and synthesized. At the same time, two restriction endonuclease sites were designed at the two ends of the template chain. The siRNA carrier insert fragment is formed by annealing. The siRNA empty vector was linearized by restriction endonuclease, and the T 4 ligase was inserted into the empty siRNA plasmid. The plasmid was identified by enzyme digestion, PCR and sequencing. The interference plasmid containing WT1 and the negative control plasmid were transferred into K562 cells by liposome. The green fluorescent protein (GFP) gene was used as the reporter gene. Flow cytometry and fluorescence microscopy were used to observe the transfection efficiency and the efficiency of plasmid expression after G418 screening for 4 weeks. RT-PCR was used to detect the expression of WT1 gene. Trypan blue exclusion assay and MTT colorimetry were used to detect the expression of WT1 gene. The effect of WT1 gene interference on the growth of K562 cells was detected by colony forming assay of methylcellulose. The effect of WT1 gene interference on apoptosis of K562 cells was observed by flow cytometry (FCM). Results 1 the average efficiency of liposome transfection into MCF-7 cells was 98.1% (97.2n 98.7%). Of the 8 pairs of siRNA designed, 4 pairs could inhibit the expression of WT1 gene. The inhibitory efficiency was between 17.3% and 89.79%. The interference efficiency of siRNA transfected with MCF-7 cells was 91.00 and 91.980.The interference efficiency was 73.02and 77.537.530.The interference efficiency was 73.02and 42.97, respectively. The RNAi effect could be maintained by 10nmol/L concentration siRNA for at least 4 days. 3 the sensitivity of WT1 gene to the apoptosis induced by vincristine was observed by flow cytometry.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346
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