RNA干扰对小鼠腹腔活化巨噬细胞分泌TNF-α的影响

发布时间：2018-04-22 05:39

本文选题：RNA干扰 + shRNA　；参考：《中国医科大学》2007年硕士论文

【摘要】： RNA干扰对小鼠腹腔活化巨噬细胞分泌TNF-α的影响目的肿瘤坏死因子(tumor necrosis factor，TNF)分为肿瘤坏死因子α和肿瘤坏死因子β，TNF-α生物学作用有杀伤肿瘤、促进炎症反应、抗炎、抗病毒、发热反应、免疫调节等，其主要由活化的巨噬细胞、T淋巴细胞和NK细胞分泌的，在多种其它细胞中也有低表达，如成纤维细胞、平滑肌细胞和肿瘤细胞。适量的TNF-α可以调节机体免疫功能，对维持机体内部的稳定状态及抵御各种致病因子具有重要意义，然而，TNF-α的异常分泌可作为机体炎症、损伤及休克的重要炎症介质。研究已证明TNF-α不仅对肿瘤细胞具有细胞毒性，还可以诱导产生多型白细胞介素和干扰素，这些细胞因子的网络活性可以相互协同，，形成一系列连锁放大反应，加重炎症反应，最终导致机体的广泛损伤，造成诸如感染性休克、多器官损害、DIC等危重症的发生。TNF-α还具有对骨组织的吸收作用而造成对骨的破坏。目前己知与TNF-α有关的疾病包括：AIDS、贫血、肿瘤、出血性休克、移植排斥反应、结核病、白血病、糖尿病、类风湿性关节炎等。TNF-α参与了众多疾病的发生发展过程，因此在不同水平上阻断TNF-α，有可能对与TNF-α有关的疾病产生治疗作用。RNA干扰(RNA interference，RNAi)是利用具有同源性互补序列的双链RNA来诱发序列特异性的转录后基因沉默现象，它可以通过抑制蛋白表达来模拟基因敲除技术，RNA干扰在植物、真菌及动物细胞中均存在，在抗肿瘤、抗病毒及研究基因功能等方面都有着广泛的应用前景。RNAi可以通过人为的引入与内源靶基因具有相同序列的双链RNA，诱导内源靶基因mRNA降解，达到阻止基因表达的目的。所以RNAi可以作为一种简单有效的可代替基因敲除技术来抑制特定基因表达的有力遗传工具，RNAi技术作为新兴的基因阻断技术具有明显优势，它具有高度特异性，靶基因中一个碱基的改变就会使RNAi效率大幅下降，因此它可以使等位基因特异性沉默；它的高效性可使靶基因表达的蛋白量下降90％以上，它比反义核酸技术有效，比基因敲除技术简单，已经迅速广泛地应用到基因功能研究、基因表达调控机制和抗病毒感染等热门领域，并为基因治疗开辟了全新的途径。本试验利用silentGene-2 U6 Hairpin cloning Systems在体外经PCR合成siRNA表达框架，利用脂质体转染入细胞后，转录出shRNA引发干扰。针对小鼠TNF-α基因选取2个靶位点，测定并比较其干扰效果，从而选出敏感的抑制位点，为今后克隆质粒提供最强的干扰序列。本文就位点1干扰作用加以论述。方法 1、小鼠腹腔巨噬细胞培养：C57BL／6小鼠，雄性(中国医科大学动物部提供)，腹腔注射2m110／1淀粉溶液，24小时后，颈椎脱臼法处死，无菌条件下，剪开腹腔，用4mlHank's液冲洗腹腔，收集冲洗液于离心管中，4℃离心，1000rpm，7min。弃上清，用无菌PBS洗细胞，4℃离心，1000rpm，7min，弃上清，用完全DMEM培养液调整细胞浓度，2×10~6个／ml，接种于6孔板。 2、巨噬细胞TNF-α产生能力的监测：细胞正常生长后，每孔加10μg／mlLPS10μl，分别于刺激2小时、6小时、10小时、14小时检测其TNF-α的分泌量。 3、实验分组：共分3组。干扰组：给予针对TNF-α干扰位点的siRNA表达框架。阴性对照组：给予正常DMEM培养液。阳性对照组：给予针对非同源siRNA表达框架，这里选择的是靶向IL-1某一位点的序列。 4、干扰组及阳性对照组siRNA表达框架的获得及检测：从GeneBank中查找C57BL／6小鼠TNF-α、IL-1的序列。根据靶序列筛选原则及Promega公司shRNAtarget Disigner设计软件，经BLAST对比后，选择出编码靶标RNA的基因序列，由上海生工公司合成干扰组及阳性对照组下游引物，利用silentGene-2 U6 Hairpincloning Systems经PCR反应合成siRNA表达框架，纯化后，用2％琼脂糖凝胶电泳分析。 5、RNA干扰作用特异性的测定：IL-1为淋巴细胞分泌的另一重要的细胞因子，将其作为阳性对照，可说明RNAi作用的特异性。 6、TNF-αmRNA的检测：收集细胞，提取RNA，用RT-PCR方法检测TNF-αmRNA，操作步骤严格按照说明书进行。 7、细胞因子的检测：干扰组、阳性对照组、阴性对照组取细胞培养液上清，用ELISA方法检测TNF-α、IL-1的分泌情况，操作严格按照说明书进行。结果 1、LPS刺激腹腔巨噬细胞产生TNF-α量的动态变化：LPS刺激后，2小时TNF-α的分泌量开始上升，6小时表达达到高峰，10小时以后开始下降。 2、干扰组及阳性对照组siRNA表达框架的获得及检测：干扰组下游引物序列： 5'ATCTAAAAAGAGCACAGAAAGCATGATCAGAGAACTTGATCATGCTTTCTGTGCTCGGTGTTTCGTCCTTTCCACAAGA3' 阳性对照组下游引物： 5'ATCTAAAAAGCAAGCTATGGCTCATTCAGAGAACTTGAATGAGCCATAGCTTGCGGTGTTTCGTCCTTTCCACAAGA3' 引物合成后，利用silentGene-2 U6 Hairpin cloning Systems经PCR反应合成siRNA表达框架。 2％琼脂糖电泳检测纯化后的PCR产物见图4。 3、RNA干扰作用对TNF-αmRNA水平的影响：干扰组TNF-αmRNA量较阳性对照组及阴性对照组均明显降低。P＜0.05。 4、RNA干扰作用序列特异性：阳性对照组IL-1分泌量较干扰组及阴性对照组均明显降低。P＜0.05。 5、RNA干扰作用对TNF-α浓度的影响：干扰组TNF-α分泌量较阳性对照组及阴性对照组均明显降低。P＜0.05。结论 1、RNA干扰可以有效地抑制原代培养小鼠腹腔巨噬细胞TNF-αmRNA水平及其蛋白的分泌量。 2、RNA干扰有严格的序列特异性。 3、针对位点1的干扰作用可以抑制小鼠腹腔巨噬细胞TNF-αmRNA的表达，该位点可以作为今后克隆质粒研究的敏感位点。总之，RNA干扰特异性抑制了哺乳动物腹腔巨噬细胞TNF-αmRNA的表达，为TNF-α相关疾病在基因水平上的治疗提供了可靠的依据。
[Abstract]:Effects of RNA interference on TNF - 伪 secretion in peritoneal activated macrophages of mice

Purpose

Tumor necrosis factor ( TNF ) is divided into tumor necrosis factor alpha ( TNF - 伪 ) and tumor necrosis factor ( TNF - 伪 ) , tumor necrosis factor - 伪 ( TNF - 伪 ) , tumor necrosis factor beta ( TNF - 伪 ) , tumor necrosis factor - 伪 ( TNF - 伪 ) , 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 - 伪 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 - 伪 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 - 伪 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 - 伪 mRNA : Cells were collected , RNA was extracted , TNF - 伪 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 - 伪 and IL - 1 , and the operation was carried out in strict accordance with the instructions .

Results

1 . LPS stimulated the dynamic changes of TNF - 伪 in peritoneal macrophages : after LPS stimulation , TNF - 伪 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 - 伪 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 - 伪 concentration : TNF - 伪 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 - 伪 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 - 伪 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 - 伪 mRNA in peritoneal macrophages of mammals , and provides a reliable basis for the treatment of TNF - 伪 related diseases at gene level .

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

【共引文献】