RNA干擾對WT1基因表達的抑制作用
發(fā)布時間：2019-02-15 10:09
【摘要】：目的研究siRNA(small interfering RNA,siRNA)對WT1基因的抑制作用,構(gòu)建WT1基因siRNA載體,為進一步探討WT1在白血病中的生物學(xué)功能及白血病的基因治療奠定基礎(chǔ)。 方法設(shè)計制備多對針對WT1基因的小干擾RNA(small interfering RNA, siRNA),通過陽離子脂質(zhì)體Lipofectamine 2000轉(zhuǎn)染乳腺癌細胞株MCF-7細胞,流式細胞儀檢測轉(zhuǎn)染效率,采用實時定量PCR(Real-time Quantitative Polymerase Chain Reaction, RQ-PCR)法測定WT1及內(nèi)參β-actin mRNA的表達,篩選出最有效的siRNA,Western Blot測定WT1蛋白的表達。用不同濃度的最有效的WT1siRNA轉(zhuǎn)染MCF-7細胞,觀察不同濃度siRNA對RNAi效應(yīng)的影響。用10nmol/L濃度的最有效siRNA轉(zhuǎn)染MCF-7細胞后,繼續(xù)培養(yǎng)24、48、72、96和120h收集細胞進行檢測分析,觀察RNAi效應(yīng)的持續(xù)時間。并用流式細胞儀(FCM)檢測WT1被干擾后,MCF-7細胞對長春新堿誘導(dǎo)凋亡的敏感性。根據(jù)最有效的siRNA序列,設(shè)計合成兩條shRNA(small hair RNA, shRNA)的DNA模板單鏈,同時模板鏈兩端分別設(shè)計不同的兩個限制酶切位點。退火形成siRNA載體插入片斷。用限制性內(nèi)切酶將siRNA空載體線性化,T4連接酶將插入片斷插入siRNA空質(zhì)粒中。經(jīng)酶切、PCR和測序的方法鑒定質(zhì)粒是否成功。通過脂質(zhì)體將裝有WT1的干擾質(zhì)粒和陰性對照質(zhì)粒轉(zhuǎn)入K562細胞中,以綠色熒光蛋白(GFP)基因為報告基因,用流式細胞儀和熒光顯微鏡觀察轉(zhuǎn)染效率和G418篩選4周后質(zhì)粒表達效率。RT-PCR檢測WT1基因表達變化,采用臺盼蘭拒染法、MTT比色法、甲基纖維素集落形成實驗檢測WT1基因干擾后對K562細胞生長的影響。通過流式細胞儀測定AnnexinV結(jié)合力觀察WT1基因干擾后對K562細胞誘導(dǎo)凋亡的作用。 結(jié)果①脂質(zhì)體轉(zhuǎn)染MCF-7細胞的效率均值為98.1%(97.2～98.7%),所設(shè)計的8對siRNA中,4對能抑制WT1基因的表達,抑制效率在17.3%~89.79%之間。②5、10、50、100、150和200nmol/L濃度的siRNA轉(zhuǎn)染MCF-7細胞后,干擾效率分別為91.00%、91.98%、81.52%、73.02%、77.53%、42.97%;在10nmol/L濃度siRNA作用下,可維持RNAi效應(yīng)至少4天時間。③流式細胞儀測定Annexin V結(jié)合力的方法觀察WT1基因被干擾后對長春新堿的誘導(dǎo)凋亡作用的敏感性,對
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R346
本文編號：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R346
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