RNA干扰在体外、体内抑制TNF-α表达的实验研究
发布时间：2019-02-21 21:31
【摘要】： 第一部分化学合成的siRNA对活化的小鼠巨噬细胞肿瘤坏死因子α表达的影响 目的观察靶向小鼠肿瘤坏死因子α(TNF-α)基因的化学合成的小干扰RNA(siRNA)在体外对小鼠巨噬细胞系RAW264.7表达TNF-α的抑制作用。方法采用化学法合成针对TNF-αmRNA不同位点设计的3条siRNA序列(siRNA1-3)和1条带有荧光标记的BLOCK-ITTM Fluorescent荧光Oligo(修饰的荧光标记的dsRNA)通过脂质体包裹后将其分别转染至小鼠巨噬细胞系RAW264.7,同时设立一个无任何靶基因的siRNA做为阴性对照(siRNA4)。荧光显微镜下观察siRNA的转染效率;用实时荧光定量PCR和酶联免疫吸附实验(ELISA)法分别检测siRNA对TNF-α的mRNA和蛋白表达的抑制作用。结果内毒素刺激后6 h ,巨噬细胞表达TNF-αmRNA和合成分泌的TNF-α量均增加,于9～12 h达高峰。利用荧光标记的Oligo观察到siRNA转染效率达72%～80%。siRNA1-4转染巨噬细胞后, siRNA2、3可见内毒素刺激的TNF-αmRNA(0.158±0.030、0.114±0.028)和TNF-α蛋白表达[(1355±348)pg/ml、(817±138)pg/ml]均明显少于未转染组[TNF-αmRNA 0.294±0.147,蛋白(2104±32)pg/ml,均P 0.05],其中siRNA3的抑制率非常显著,达61.2%(P 0.01)。siRNA1、阴性对照siRNA4对细胞基因及蛋白表达无影响。结论内毒素可刺激小鼠巨噬细胞TNF-α的合成。化学合成的siRNA转染小鼠巨噬细胞能有效抑制TNF-αmRNA及蛋白的表达。 第二部分发夹样siRNA抑制小鼠巨噬细胞TNF-α表达 目的:研究特异性载体介导的小干扰RNA(siRNA)对小鼠巨噬细胞株RAW264.7表达肿瘤坏死因子α(TNF-α)基因的抑制作用。方法:设计两段靶向TNF-α基因的特异性siRNA,合成相应寡核苷酸,插入载体H1启动子下游,构建表达干扰性发夹状RNA(Short hairpin RNA, shRNA)的载体pHS-A和pHS-B,转染小鼠巨噬细胞株,用实时荧光定量PCR和ELISA法检测siRNA对TNF-α的mRNA和蛋白表达的抑制作用。 结果: pHS-A转染后,LPS刺激巨噬细胞的TNF-αmRNA为0.00356±0.00187, TNF-α蛋白表达为149.93±21.02 pg/ml,比未转染组(TNF-αmRNA 0.02134±0.00960,蛋白1922.30±149.05pg/ml)显著减少P 0.01),抑制率达83.3%。pHS-B及阴性对照组对基因及蛋白表达均无影响。结论: pHS-A可成功地表达了发夹状,并且能抑制小鼠巨噬细胞TNF-α表达。 第三部分应用载体构建shRNA对小鼠葡聚糖硫酸钠结肠炎的影响 目的:探讨载体构建shRNA对小鼠葡聚糖硫酸钠( DSS)结肠炎的治疗作用,寻找治疗溃疡性结肠炎(UC)的新途径。方法: 30只BALB /C小鼠随机分为: (1)生理盐水对照组(2)4% DSS口服+生理盐水模型组(3)4% DSS口服+载体构建shRNA治疗组,每组10只。各组小鼠每天记录和计算其结肠炎活动指数(DAI)评分;取小鼠结肠粘膜组织进行大体及组织形态学观察、用酶联免疫吸附法( ELISA)测定肿瘤坏死因子 α(TNF α)水平、荧光定量PCR检测TNF-αmRNA表达。结果:模型组和治疗组小鼠的DAI评分、组织学评分和肠粘膜组织内TNF-α基因和蛋白表达均明显高于正常对照组(均P 0. O5);而治疗组的上述指标均又明显低于模型组,治疗组与对照组的结肠组织学评分和TNF-αTNF-α基因和蛋白的表达分别为5.30±0.48和8.50±0.53(P 0.05),0.31±0.11和0.42±0.04(P 0.05),(28.13±3.23)pg/g和(39.43±3.90)pg/g(P 0.05)。结论:载体构建的shRNA对小鼠DSS结肠炎有保护作用。
[Abstract]:The effect of chemically synthesized siRNA on the expression of tumor necrosis factor in activated mouse macrophages Objective To observe the effect of small interfering RNA (siRNA) on the expression of TNF-1 in mouse macrophage system RAW264.7 in vitro. Three siRNA sequences (siRNAs 1-3) designed for different sites of TNF-1 mRNA and 1 with fluorescent-labeled BLOCK-ITTM Fluoriferous fluorescent Oleno (modified fluorescent labeled dsRNA) were synthesized by chemical method, and then transfected into mouse macrophage system RAW26 after being wrapped by liposome. 4.7. Establish a siRNA without any target gene as the negative control (siRN A4) The transfection efficiency of siRNA was observed under a fluorescence microscope, and the expression of the mRNA and protein of TNF-1 was detected by real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay (ELISA). The expression of TNF-1 mRNA and the amount of TNF-1 secreted by macrophages increased at 6 h after endotoxemia and 9-12 h. When the transfection efficiency of siRNA1-4 was 72% ~ 80%. After siRNA1-4 was transfected into macrophages, the TNF-1 mRNA (0.158-0.030, 0.114-028) and the expression of TNF-linked protein[(1355-348) pg/ ml, (817-138) pg/ ml] were significantly less than that of the non-transfected group[TNF-1 mRNA 0. 294] 0. 147, protein (2104-32) pg/ ml, all P 0.05], wherein the inhibition rate of siRNA3 was very significant, up to 61.2% (P 0). 01). siRNA1, negative control siRNA4 for cell gene and protein expression No effect. Conclusion Endotoxin can stimulate the TNF-1 of mouse macrophages. The synthetic and chemical synthesis of siRNA-transfected mouse macrophages can effectively inhibit the TNF-1 mRNA and protein. Expression of the second part of hairpin-like siRNA in the inhibition of macrophagocytosis in mice Objective: To study the expression of tumor necrosis factor (TN) in mouse macrophage strain RAW264.7 by using specific vector-mediated small interfering RNA (siRNA). and a vector pHS-A and pHS-A for expressing the interfering hairpin RNA (shRNA) are constructed downstream of the promoter H1 promoter to construct a vector pHS-A and pHS-A expressing the interfering hairpin RNA (shRNA). B, the mouse macrophage strain is transfected, and the mRN of the siRNA to the TNF-1 is detected by the real-time fluorescence quantitative PCR and the ELISA method Results: After pHS-A was transfected with pHS-A, the TNF-1 mRNA of LPS-stimulated macrophages was 0.35356-0.00187, and the expression of TNF-1 protein was 149.93-21.02 pg/ ml, which was lower than that of untransfected group (TNF-1 mRNA) 0.02134-0. 00960, protein 1922. 30-149.05p. g/ ml significantly reduced P 0.01) with an inhibition rate of 83.3%. pHS-B and negative The control group had no effect on the expression of gene and protein. Conclusion: pHS-A can successfully express the hairpin shape and can Inhibition of the expression of TNF-antigen in mouse macrophages. The third part of the application of vector construction Objective: To investigate the effect of shRNA on the colitis of dextran sodium sulfate in mice: to investigate the effect of shRNA on the colitis of the mouse dextran sulfate (DSS) Methods: 30 BALB/ C mice were randomly divided into: (1) normal saline control group (2) 4% DSS oral + physiological saline model group (3) 4% D SS鍙ｆ湇+杞戒綋鏋勫缓shRNA娌荤枟缁，
本文编号：2427912
[Abstract]:The effect of chemically synthesized siRNA on the expression of tumor necrosis factor in activated mouse macrophages Objective To observe the effect of small interfering RNA (siRNA) on the expression of TNF-1 in mouse macrophage system RAW264.7 in vitro. Three siRNA sequences (siRNAs 1-3) designed for different sites of TNF-1 mRNA and 1 with fluorescent-labeled BLOCK-ITTM Fluoriferous fluorescent Oleno (modified fluorescent labeled dsRNA) were synthesized by chemical method, and then transfected into mouse macrophage system RAW26 after being wrapped by liposome. 4.7. Establish a siRNA without any target gene as the negative control (siRN A4) The transfection efficiency of siRNA was observed under a fluorescence microscope, and the expression of the mRNA and protein of TNF-1 was detected by real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay (ELISA). The expression of TNF-1 mRNA and the amount of TNF-1 secreted by macrophages increased at 6 h after endotoxemia and 9-12 h. When the transfection efficiency of siRNA1-4 was 72% ~ 80%. After siRNA1-4 was transfected into macrophages, the TNF-1 mRNA (0.158-0.030, 0.114-028) and the expression of TNF-linked protein[(1355-348) pg/ ml, (817-138) pg/ ml] were significantly less than that of the non-transfected group[TNF-1 mRNA 0. 294] 0. 147, protein (2104-32) pg/ ml, all P 0.05], wherein the inhibition rate of siRNA3 was very significant, up to 61.2% (P 0). 01). siRNA1, negative control siRNA4 for cell gene and protein expression No effect. Conclusion Endotoxin can stimulate the TNF-1 of mouse macrophages. The synthetic and chemical synthesis of siRNA-transfected mouse macrophages can effectively inhibit the TNF-1 mRNA and protein. Expression of the second part of hairpin-like siRNA in the inhibition of macrophagocytosis in mice Objective: To study the expression of tumor necrosis factor (TN) in mouse macrophage strain RAW264.7 by using specific vector-mediated small interfering RNA (siRNA). and a vector pHS-A and pHS-A for expressing the interfering hairpin RNA (shRNA) are constructed downstream of the promoter H1 promoter to construct a vector pHS-A and pHS-A expressing the interfering hairpin RNA (shRNA). B, the mouse macrophage strain is transfected, and the mRN of the siRNA to the TNF-1 is detected by the real-time fluorescence quantitative PCR and the ELISA method Results: After pHS-A was transfected with pHS-A, the TNF-1 mRNA of LPS-stimulated macrophages was 0.35356-0.00187, and the expression of TNF-1 protein was 149.93-21.02 pg/ ml, which was lower than that of untransfected group (TNF-1 mRNA) 0.02134-0. 00960, protein 1922. 30-149.05p. g/ ml significantly reduced P 0.01) with an inhibition rate of 83.3%. pHS-B and negative The control group had no effect on the expression of gene and protein. Conclusion: pHS-A can successfully express the hairpin shape and can Inhibition of the expression of TNF-antigen in mouse macrophages. The third part of the application of vector construction Objective: To investigate the effect of shRNA on the colitis of dextran sodium sulfate in mice: to investigate the effect of shRNA on the colitis of the mouse dextran sulfate (DSS) Methods: 30 BALB/ C mice were randomly divided into: (1) normal saline control group (2) 4% DSS oral + physiological saline model group (3) 4% D SS鍙ｆ湇+杞戒綋鏋勫缓shRNA娌荤枟缁，
本文编号：2427912
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