【摘要】： 第一部分化學(xué)合成的siRNA對(duì)活化的小鼠巨噬細(xì)胞腫瘤壞死因子α表達(dá)的影響 目的觀察靶向小鼠腫瘤壞死因子α(TNF-α)基因的化學(xué)合成的小干擾RNA(siRNA)在體外對(duì)小鼠巨噬細(xì)胞系RAW264.7表達(dá)TNF-α的抑制作用。方法采用化學(xué)法合成針對(duì)TNF-αmRNA不同位點(diǎn)設(shè)計(jì)的3條siRNA序列(siRNA1-3)和1條帶有熒光標(biāo)記的BLOCK-ITTM Fluorescent熒光Oligo(修飾的熒光標(biāo)記的dsRNA)通過脂質(zhì)體包裹后將其分別轉(zhuǎn)染至小鼠巨噬細(xì)胞系RAW264.7,同時(shí)設(shè)立一個(gè)無任何靶基因的siRNA做為陰性對(duì)照(siRNA4)。熒光顯微鏡下觀察siRNA的轉(zhuǎn)染效率;用實(shí)時(shí)熒光定量PCR和酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)法分別檢測(cè)siRNA對(duì)TNF-α的mRNA和蛋白表達(dá)的抑制作用。結(jié)果內(nèi)毒素刺激后6 h ,巨噬細(xì)胞表達(dá)TNF-αmRNA和合成分泌的TNF-α量均增加,于9～12 h達(dá)高峰。利用熒光標(biāo)記的Oligo觀察到siRNA轉(zhuǎn)染效率達(dá)72%～80%。siRNA1-4轉(zhuǎn)染巨噬細(xì)胞后, siRNA2、3可見內(nèi)毒素刺激的TNF-αmRNA(0.158±0.030、0.114±0.028)和TNF-α蛋白表達(dá)[(1355±348)pg/ml、(817±138)pg/ml]均明顯少于未轉(zhuǎn)染組[TNF-αmRNA 0.294±0.147,蛋白(2104±32)pg/ml,均P 0.05],其中siRNA3的抑制率非常顯著,達(dá)61.2%(P 0.01)。siRNA1、陰性對(duì)照siRNA4對(duì)細(xì)胞基因及蛋白表達(dá)無影響。結(jié)論內(nèi)毒素可刺激小鼠巨噬細(xì)胞TNF-α的合成。化學(xué)合成的siRNA轉(zhuǎn)染小鼠巨噬細(xì)胞能有效抑制TNF-αmRNA及蛋白的表達(dá)。 第二部分發(fā)夾樣siRNA抑制小鼠巨噬細(xì)胞TNF-α表達(dá) 目的:研究特異性載體介導(dǎo)的小干擾RNA(siRNA)對(duì)小鼠巨噬細(xì)胞株RAW264.7表達(dá)腫瘤壞死因子α(TNF-α)基因的抑制作用。方法:設(shè)計(jì)兩段靶向TNF-α基因的特異性siRNA,合成相應(yīng)寡核苷酸,插入載體H1啟動(dòng)子下游,構(gòu)建表達(dá)干擾性發(fā)夾狀RNA(Short hairpin RNA, shRNA)的載體pHS-A和pHS-B,轉(zhuǎn)染小鼠巨噬細(xì)胞株,用實(shí)時(shí)熒光定量PCR和ELISA法檢測(cè)siRNA對(duì)TNF-α的mRNA和蛋白表達(dá)的抑制作用。 結(jié)果: pHS-A轉(zhuǎn)染后,LPS刺激巨噬細(xì)胞的TNF-αmRNA為0.00356±0.00187, TNF-α蛋白表達(dá)為149.93±21.02 pg/ml,比未轉(zhuǎn)染組(TNF-αmRNA 0.02134±0.00960,蛋白1922.30±149.05pg/ml)顯著減少P 0.01),抑制率達(dá)83.3%。pHS-B及陰性對(duì)照組對(duì)基因及蛋白表達(dá)均無影響。結(jié)論: pHS-A可成功地表達(dá)了發(fā)夾狀,并且能抑制小鼠巨噬細(xì)胞TNF-α表達(dá)。 第三部分應(yīng)用載體構(gòu)建shRNA對(duì)小鼠葡聚糖硫酸鈉結(jié)腸炎的影響 目的:探討載體構(gòu)建shRNA對(duì)小鼠葡聚糖硫酸鈉( DSS)結(jié)腸炎的治療作用,尋找治療潰瘍性結(jié)腸炎(UC)的新途徑。方法: 30只BALB /C小鼠隨機(jī)分為: (1)生理鹽水對(duì)照組(2)4% DSS口服+生理鹽水模型組(3)4% DSS口服+載體構(gòu)建shRNA治療組,每組10只。各組小鼠每天記錄和計(jì)算其結(jié)腸炎活動(dòng)指數(shù)(DAI)評(píng)分;取小鼠結(jié)腸粘膜組織進(jìn)行大體及組織形態(tài)學(xué)觀察、用酶聯(lián)免疫吸附法( ELISA)測(cè)定腫瘤壞死因子 α(TNF α)水平、熒光定量PCR檢測(cè)TNF-αmRNA表達(dá)。結(jié)果:模型組和治療組小鼠的DAI評(píng)分、組織學(xué)評(píng)分和腸粘膜組織內(nèi)TNF-α基因和蛋白表達(dá)均明顯高于正常對(duì)照組(均P 0. O5);而治療組的上述指標(biāo)均又明顯低于模型組,治療組與對(duì)照組的結(jié)腸組織學(xué)評(píng)分和TNF-αTNF-α基因和蛋白的表達(dá)分別為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)。結(jié)論:載體構(gòu)建的shRNA對(duì)小鼠DSS結(jié)腸炎有保護(hù)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+杞戒綋鏋勫緩shRNA娌葷枟緇，

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