本文選題：RNA干擾 + shRNA��； 參考：《中國醫(yī)科大學(xué)》2007年碩士論文

【摘要】： RNA干擾對小鼠腹腔活化巨噬細(xì)胞分泌TNF-α的影響 目的 腫瘤壞死因子(tumor necrosis factor，TNF)分為腫瘤壞死因子α和腫瘤壞死因子β，TNF-α生物學(xué)作用有殺傷腫瘤、促進(jìn)炎癥反應(yīng)、抗炎、抗病毒、發(fā)熱反應(yīng)、免疫調(diào)節(jié)等，其主要由活化的巨噬細(xì)胞、T淋巴細(xì)胞和NK細(xì)胞分泌的，在多種其它細(xì)胞中也有低表達(dá)，如成纖維細(xì)胞、平滑肌細(xì)胞和腫瘤細(xì)胞。適量的TNF-α可以調(diào)節(jié)機(jī)體免疫功能，對維持機(jī)體內(nèi)部的穩(wěn)定狀態(tài)及抵御各種致病因子具有重要意義，然而，TNF-α的異常分泌可作為機(jī)體炎癥、損傷及休克的重要炎癥介質(zhì)。研究已證明TNF-α不僅對腫瘤細(xì)胞具有細(xì)胞毒性，還可以誘導(dǎo)產(chǎn)生多型白細(xì)胞介素和干擾素，這些細(xì)胞因子的網(wǎng)絡(luò)活性可以相互協(xié)同，，形成一系列連鎖放大反應(yīng)，加重炎癥反應(yīng)，最終導(dǎo)致機(jī)體的廣泛損傷，造成諸如感染性休克、多器官損害、DIC等危重癥的發(fā)生。TNF-α還具有對骨組織的吸收作用而造成對骨的破壞。目前己知與TNF-α有關(guān)的疾病包括：AIDS、貧血、腫瘤、出血性休克、移植排斥反應(yīng)、結(jié)核病、白血病、糖尿病、類風(fēng)濕性關(guān)節(jié)炎等。TNF-α參與了眾多疾病的發(fā)生發(fā)展過程，因此在不同水平上阻斷TNF-α，有可能對與TNF-α有關(guān)的疾病產(chǎn)生治療作用。RNA干擾(RNA interference，RNAi)是利用具有同源性互補(bǔ)序列的雙鏈RNA來誘發(fā)序列特異性的轉(zhuǎn)錄后基因沉默現(xiàn)象，它可以通過抑制蛋白表達(dá)來模擬基因敲除技術(shù)，RNA干擾在植物、真菌及動物細(xì)胞中均存在，在抗腫瘤、抗病毒及研究基因功能等方面都有著廣泛的應(yīng)用前景。RNAi可以通過人為的引入與內(nèi)源靶基因具有相同序列的雙鏈RNA，誘導(dǎo)內(nèi)源靶基因mRNA降解，達(dá)到阻止基因表達(dá)的目的。所以RNAi可以作為一種簡單有效的可代替基因敲除技術(shù)來抑制特定基因表達(dá)的有力遺傳工具，RNAi技術(shù)作為新興的基因阻斷技術(shù)具有明顯優(yōu)勢，它具有高度特異性，靶基因中一個堿基的改變就會使RNAi效率大幅下降，因此它可以使等位基因特異性沉默；它的高效性可使靶基因表達(dá)的蛋白量下降90％以上，它比反義核酸技術(shù)有效，比基因敲除技術(shù)簡單，已經(jīng)迅速廣泛地應(yīng)用到基因功能研究、基因表達(dá)調(diào)控機(jī)制和抗病毒感染等熱門領(lǐng)域，并為基因治療開辟了全新的途徑。本試驗(yàn)利用silentGene-2 U6 Hairpin cloning Systems在體外經(jīng)PCR合成siRNA表達(dá)框架，利用脂質(zhì)體轉(zhuǎn)染入細(xì)胞后，轉(zhuǎn)錄出shRNA引發(fā)干擾。針對小鼠TNF-α基因選取2個靶位點(diǎn)，測定并比較其干擾效果，從而選出敏感的抑制位點(diǎn)，為今后克隆質(zhì)粒提供最強(qiáng)的干擾序列。本文就位點(diǎn)1干擾作用加以論述。 方法 1、小鼠腹腔巨噬細(xì)胞培養(yǎng)：C57BL／6小鼠，雄性(中國醫(yī)科大學(xué)動物部提供)，腹腔注射2m110／1淀粉溶液，24小時后，頸椎脫臼法處死，無菌條件下，剪開腹腔，用4mlHank's液沖洗腹腔，收集沖洗液于離心管中，4℃離心，1000rpm，7min。棄上清，用無菌PBS洗細(xì)胞，4℃離心，1000rpm，7min，棄上清，用完全DMEM培養(yǎng)液調(diào)整細(xì)胞濃度，2×10~6個／ml，接種于6孔板。 2、巨噬細(xì)胞TNF-α產(chǎn)生能力的監(jiān)測：細(xì)胞正常生長后，每孔加10μg／mlLPS10μl，分別于刺激2小時、6小時、10小時、14小時檢測其TNF-α的分泌量。 3、實(shí)驗(yàn)分組：共分3組。干擾組：給予針對TNF-α干擾位點(diǎn)的siRNA表達(dá)框架。陰性對照組：給予正常DMEM培養(yǎng)液。陽性對照組：給予針對非同源siRNA表達(dá)框架，這里選擇的是靶向IL-1某一位點(diǎn)的序列。 4、干擾組及陽性對照組siRNA表達(dá)框架的獲得及檢測：從GeneBank中查找C57BL／6小鼠TNF-α、IL-1的序列。根據(jù)靶序列篩選原則及Promega公司shRNAtarget Disigner設(shè)計(jì)軟件，經(jīng)BLAST對比后，選擇出編碼靶標(biāo)RNA的基因序列，由上海生工公司合成干擾組及陽性對照組下游引物，利用silentGene-2 U6 Hairpincloning Systems經(jīng)PCR反應(yīng)合成siRNA表達(dá)框架，純化后，用2％瓊脂糖凝膠電泳分析。 5、RNA干擾作用特異性的測定：IL-1為淋巴細(xì)胞分泌的另一重要的細(xì)胞因子，將其作為陽性對照，可說明RNAi作用的特異性。 6、TNF-αmRNA的檢測：收集細(xì)胞，提取RNA，用RT-PCR方法檢測TNF-αmRNA，操作步驟嚴(yán)格按照說明書進(jìn)行。 7、細(xì)胞因子的檢測：干擾組、陽性對照組、陰性對照組取細(xì)胞培養(yǎng)液上清，用ELISA方法檢測TNF-α、IL-1的分泌情況，操作嚴(yán)格按照說明書進(jìn)行。 結(jié)果 1、LPS刺激腹腔巨噬細(xì)胞產(chǎn)生TNF-α量的動態(tài)變化：LPS刺激后，2小時TNF-α的分泌量開始上升，6小時表達(dá)達(dá)到高峰，10小時以后開始下降。 2、干擾組及陽性對照組siRNA表達(dá)框架的獲得及檢測： 干擾組下游引物序列： 5'ATCTAAAAAGAGCACAGAAAGCATGATCAGAGAACTTGATCATGCTTTCTGTGCTCGGTGTTTCGTCCTTTCCACAAGA3' 陽性對照組下游引物： 5'ATCTAAAAAGCAAGCTATGGCTCATTCAGAGAACTTGAATGAGCCATAGCTTGCGGTGTTTCGTCCTTTCCACAAGA3' 引物合成后，利用silentGene-2 U6 Hairpin cloning Systems經(jīng)PCR反應(yīng)合成siRNA表達(dá)框架。 2％瓊脂糖電泳檢測純化后的PCR產(chǎn)物見圖4。 3、RNA干擾作用對TNF-αmRNA水平的影響：干擾組TNF-αmRNA量較陽性對照組及陰性對照組均明顯降低。P＜0.05。 4、RNA干擾作用序列特異性：陽性對照組IL-1分泌量較干擾組及陰性對照組均明顯降低。P＜0.05。 5、RNA干擾作用對TNF-α濃度的影響：干擾組TNF-α分泌量較陽性對照組及陰性對照組均明顯降低。P＜0.05。 結(jié)論 1、RNA干擾可以有效地抑制原代培養(yǎng)小鼠腹腔巨噬細(xì)胞TNF-αmRNA水平及其蛋白的分泌量。 2、RNA干擾有嚴(yán)格的序列特異性。 3、針對位點(diǎn)1的干擾作用可以抑制小鼠腹腔巨噬細(xì)胞TNF-αmRNA的表達(dá)，該位點(diǎn)可以作為今后克隆質(zhì)粒研究的敏感位點(diǎn)。 總之，RNA干擾特異性抑制了哺乳動物腹腔巨噬細(xì)胞TNF-αmRNA的表達(dá)，為TNF-α相關(guān)疾病在基因水平上的治療提供了可靠的依據(jù)。
[Abstract]:Effects of RNA interference on TNF - 偽 secretion in peritoneal activated macrophages of mice






Purpose






Tumor necrosis factor ( TNF ) is divided into tumor necrosis factor alpha ( TNF - 偽 ) and tumor necrosis factor ( TNF - 偽 ) , tumor necrosis factor - 偽 ( TNF - 偽 ) , tumor necrosis factor beta ( TNF - 偽 ) , tumor necrosis factor - 偽 ( TNF - 偽 ) , anti - inflammatory , anti - virus , fever response and immune regulation . So RNAi can be used as a simple and effective genetic tool which can replace gene knockout technology to inhibit the expression of specific gene . So RNAi can be used as a simple and effective gene - blocking technique to inhibit gene expression .
Its high efficiency can decrease the amount of protein expressed by target gene by more than 90 % , which is more effective than antisense nucleic acid technology . It has been widely used in gene function research , gene expression regulation mechanism and anti - virus infection .






method






1 . Mouse peritoneal macrophages were cultured : C57BL / 6 mice , male ( supplied by the Animal Department of China Medical University ) , intraperitoneal injection of 2m110 / 1 starch solution , 2 m110 / 1 starch solution were injected intraperitoneally , the abdominal cavity was washed with 4 ml Hank ' s solution , washed with 4 ml Hank ' s solution , washed with 4 ml Hank ' s solution , centrifuged at 1000 rpm for 7 min , the supernatant was discarded , the cell concentration was adjusted at 4.degree . C. , 2.times . 10 - 6 cells / ml , and inoculated to 6 - well plates .






2 . Monitoring of TNF - 偽 production ability of macrophages : After normal cell growth , 10.mu . g / ml of LPS10 . mu.l were added to each well , and TNF - . alpha . secretion was detected at 2 hours , 6 hours , 10 hours and 14 hours , respectively .






3 . Experimental group : Three groups were divided into three groups : siRNA expression framework for TNF - 偽 interference site . Negative control group : Normal DMEM culture solution was given . Positive control group : The expression framework for non - homologous siRNA was given , and the sequence of target IL - 1 was selected here .






4 . The expression framework of siRNA was obtained and tested in the interference group and the positive control group . The sequence of TNF - 偽 and IL - 1 in C57BL / 6 mice was searched from GeneBank . After BLAST comparison , the gene sequence encoding target RNA was selected , and the siRNA expression frame was synthesized by PCR reaction between the synthetic interference group and the positive control group . After purification , two % agarose gel electrophoresis was used to analyze the siRNA expression framework .






5 . Measurement of the specific effect of RNA interference : IL - 1 is another important cytokine secreted by lymphocytes , which can be used as a positive control to demonstrate the specificity of RNAi effect .






6 . Detection of TNF - 偽 mRNA : Cells were collected , RNA was extracted , TNF - 偽 mRNA was detected by RT - PCR , and the procedure was carried out in strict accordance with the instructions .






7 . Detection of cytokines : interference group , positive control group , negative control group , cell culture fluid supernatant , ELISA method to detect the secretion of TNF - 偽 and IL - 1 , and the operation was carried out in strict accordance with the instructions .






Results






1 . LPS stimulated the dynamic changes of TNF - 偽 in peritoneal macrophages : after LPS stimulation , TNF - 偽 secretion began to rise in 2 hours , peaked at 6 hours , and began to decline after 10 hours .






2 . obtaining and detecting siRNA expression framework of the interference group and the positive control group :






Downstream primer sequence of the interference group :






5 ' ATCTAAAAAGAGCACAGAAAGCATGATCAROAACTTGATCATGCTTTCTGTGCTCGGTGTTTCGTCCTTTCCACAAGA3 '






Downstream primer of positive control group :






5 ' ATCTAAAAAGCAAGCTATGGCTCATTCAAGCAAGCTATGCCATAGCTTGCGGTGTTTCGTCCTTTCCACAAGA3 '






After the primer was synthesized , the siRNA expression framework was synthesized by PCR reaction by using PCR - PCR reaction .






The PCR product after the 2 % agarose gel electrophoresis detection and purification is shown in FIG . 4 .






3 . The effects of RNA interference on the mRNA level of TNF - 偽 mRNA were significantly lower than those in the positive control group and the negative control group ( P < 0.05 ) .






4 . The specificity of RNA interference sequence : IL - 1 secretion in the positive control group was significantly lower than that in the control group and the negative control group ( P < 0.05 ) .






5 . The effect of RNA interference on TNF - 偽 concentration : TNF - 偽 secretion in the interfering group was significantly lower than that in the positive control group and the negative control group ( P < 0.05 ) .






Conclusion






1 . RNA interference can effectively inhibit the level of TNF - 偽 mRNA and the secretion of protein in the peritoneal macrophages of primary cultured mouse .






2 . RNA interference has strict sequence specificity .






3 . The interfering effect of site 1 can inhibit the expression of TNF - 偽 mRNA in peritoneal macrophages of mice , which can be used as a sensitive site for the research of cloning plasmid in the future .






In conclusion , RNA interference specifically inhibits the expression of TNF - 偽 mRNA in peritoneal macrophages of mammals , and provides a reliable basis for the treatment of TNF - 偽 related diseases at gene level .

【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R363

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Francisco Javier Guzmán-de la Garza;Carlos Rodrigo Cámara-Lemarroy;Gabriela Alarcón-Galván;Paula Cordero-Pérez;Linda Elsa Mu濼oz-Espinosa;Nancy Esthela Fernández-Garza;;Different patterns of intestinal response to injury after arterial,venous or arteriovenous occlusion in rats[J];World Journal of Gastroenterology;2009年31期

本文編號：1785872