miR-136-5p靶向调控锌指蛋白A20在大鼠星形胶质细胞炎症中的分子机制研究

发布时间：2018-01-22 10:36

本文关键词： miR-136-5p 急性脊髓损伤炎症 A20　出处：《广西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：星形胶质细胞在神经系统中广泛存在,并与神经元以及其他胶质细胞共同构成中枢神经系统。星形胶质细胞除填充以及营养神经元的作用外,亦在神经系统的发育以及修复中也占有一席之地。急性脊髓损伤(ASCI)发生后,除直接暴力所带来的原发性损伤外,多数患者在数天内将发生继发性脊髓损伤(SSCI)。原发性损伤是外力直接作用所带来的直接性物理伤害,同其他意外伤害一样无直接有效的方法可以针对损伤进行治疗;继发性脊髓损伤是直接性损伤发生后,一段时间之内因为身体免疫系统对损伤组织激活,所导致的以细胞炎性反应损伤,并最终导致直接损伤部位的加重以及范围扩大。现有的资料表明,急性脊髓损伤中,最终要的病理生理变化是因为炎性因子所导致的细胞自我凋亡,这也是急性脊髓损伤中组织损伤较原发性损伤扩大并加重的重要机制。趋化因子(chemotactic factor)是一类具有促炎和迁移作用的小分子物质。通常在炎症反应中作为免疫细胞的化学诱导物,引导免疫细胞到达炎性反应区域发挥吞噬作用。趋化因子及其受体在中枢神经系统中广泛表达,对于干细胞的迁移、轴突的生长和神经传导的调节有重要作用。例如SCI后给予XCL-10封闭抗体进行治疗可以显著降低炎症反应,增强功能的恢复、组织和神经元轴突再生。如,有研究发现,外周神经损伤后,CXCL-12(又称MIP-2)可以通过促进中枢神经系统髓鞘的生成以及神经突的生长,增强SCI后轴突再生和重建。微小RNA(Micro-RNA或mi RNA)是一类小型非编码RNA分子,可通过碱基互补配对与靶基因的3'非翻译区(3'-ntranslated region,3'-UTR)结合而进而调控编码基因的表达。大量研究表明,mi RNAs可广泛参与肿瘤形成、免疫性疾病等几乎多种疾病的病理过程。mi R-136为mi RNA家族成员之一,在以往研究中发现,mi R-136在非小细胞癌中较其他组织表达量出现上调,上调程度与肿瘤的恶性程度及是否容易发生转移密切相关。另有研究指出mi R-136可通过下调通道蛋白Smad2和Smad3抑制肺腺癌细胞的转移与侵袭,并通过下调星形胶质细胞抑制胶质瘤细胞的凋亡。A20通常也被称为TNF-α诱导蛋白3(TNFAIP3)或锌指蛋白3,可以在内皮细胞受到TNF-α刺激后产生。A20的保护作用在天然免疫、继发性免疫中均表现出明显效果,部分研究也指出A20具有抑制B细胞肿瘤的发生。另外,A20是终止激活后的NF-κB关键蛋白,并由一些受体如TNF受体、Toll样受体、核苷酸结合寡聚化结构域蛋白2的等调节。敲除A20的大鼠体内IKK活性会病态增加使NF-κB信号通路不受控制,最终因多器官炎症反应衰竭以及组织损伤而死亡。作为一种调控蛋白,A20缺失也会导致TLR信号传导的异常并加重机体自身炎症反应的程度。本实验研究白介素17(IL-17)刺激大鼠脊髓星形胶质细胞产生炎性因子,以及A20蛋白的变化;沉默大鼠星形胶质细胞中mi R-136-5p基因的表达,IL-17诱导的大鼠星形胶质细胞炎症反应中A20蛋白变化。为急性脊髓损伤的临床治疗提供理论依据和新方法。目的:探讨A20炎症抑制蛋白在炎症因子IL-17诱导大鼠星形胶质细胞炎症反应中于mi R-136-5p被抑制前后的产量变化。方法:(1)体外分离以及培养大鼠星形胶质细胞;(2)使用不同浓度的IL-17(10、20、50、100、200ng/ml)对获得的大鼠星形胶质细胞刺激12h;(3)使用上一步中获知的最适浓度,刺激不同时间点(3h、6h、12h、24h和48h)的大鼠星形胶质细胞;(4)通过q PCR以及ELISA的方法分别检测IL-17刺激之后大鼠星形胶质细胞炎症因子(IL-6、TNF-α、MIP-2、MCP-1、MCP-5)的改变情况。(5)对星形胶质细胞进行最适浓度、最适时间的刺激,并通过q PCR以及WB方法分别测量其A20的m RNA以及蛋白表达水平。(6)沉默大鼠星形胶质细胞mi R-136-5p的基因表达;然后予IL-17刺激并q PCR测定mi R-136-5p基因水平表达,WB方法检测大鼠星形胶质细胞的A20蛋白表达水平。结果:实验组大鼠星形胶质细胞受IL-17刺激后,A20蛋白在6h时表达量显著减少(p0.05),至12h时至峰值,但是与6h相比变化无统计学差异;IL-17诱导下,与空白对照组(blank)以及阴性转染组(control)比较,mi R-136-5p基因沉默组中A20的表达量显著性的增加。结论:大鼠星形胶质细胞A20的表达量相关炎症因子变化在峰值的表达量上出现了负相关性,A20炎症负调控蛋白在大鼠星形胶质细胞炎症发挥调控作用。沉默大鼠星形胶质细胞mi R-136-5p的表达之后再次进行IL-17刺激,表明mi R-136-5p与A20之间存在着负性调控作用。因此,IL-17诱导的大鼠星形胶质细胞炎症中,沉默mi R-136-5p通过上调锌指蛋白A20发挥抗炎作用。
[Abstract]:Astrocytes widely exist in the nervous system, and neurons and other glial cells constitute the central nervous system. Astrocytes and neurons besides filling the role of nutrition, but also in the development of the nervous system and also occupy a space for one person. The repair of acute spinal cord injury (ASCI) occurred, in addition to the direct violence caused by the original injuries, most patients will occur after spinal cord injury (SSCI) in a few days. The primary injury is direct physical damage caused by the external force directly, as well as other accident without a direct and effective method to treatment for injury; secondary spinal cord injury occurs directly after injury. A long time because of the body's immune system activation on tissue injury, caused by cell inflammatory injury, and eventually lead to direct injury aggravated and expand the scope of Large. Available data indicate that the acute spinal cord injury, pathological and physiological changes to the final because of inflammatory cytokines caused by cell apoptosis is an important mechanism of self, which is the acute spinal cord injury in primary injury injury was expanding and aggravating. Chemokine (chemotactic factor) is a class of small molecules proinflammatory and migration effects. Usually in inflammatory reaction as chemically induced immune cells, direct the immune cells to play the phagocytosis of inflammatory reaction area. Chemokines and their receptors are widely expressed in the central nervous system, for stem cell migration, axon growth and nerve conduction regulation plays an important role. For example, SCI was given XCL-10 blocking antibody treatment can significantly reduce the inflammatory reaction, enhance the functional recovery and axonal regeneration, tissue. For example, studies have found that peripheral nerve injury after CXCL. -12 (also called MIP-2) can promote central nervous system myelin formation and neurite growth, enhance axonal regeneration and reconstruction after SCI. Micro RNA (Micro-RNA or MI RNA) is a class of small non encoding RNA molecules through complementary base pairing with target gene 3'untranslated region (3'-ntranslated region, 3'-UTR and then combined with) the regulation of the expression of the gene encoding. A large number of studies show that MI RNAs can be widely involved in tumor formation,.Mi R-136 pathological process of immune disease almost a variety of diseases as one of the MI members of the RNA family, found in previous research, MI R-136 compared with other tissues in non-small cell carcinoma was up-regulated, malignant increased degree of tumor and is prone to metastasis. Another study pointed out that MI R-136 can transfer and invasion down channel protein Smad2 and Smad3 inhibit the lung adenocarcinoma cells, and through the cut .A20 glioma cell apoptosis inhibition of astrocytes is often referred to as TNF- alpha induced protein 3 (TNFAIP3) or zinc finger protein 3, which can be stimulated with TNF- in endothelial cells to produce the protective effect of.A20 in innate immunity and secondary immune showed obvious effect, part of the study also pointed out that A20 has inhibition of B cell tumor. In addition, A20 is the termination of NF- kappa B key protein after activation, and by some receptors such as TNF receptor, Toll like receptor, nucleotide binding oligomerization domain protein 2. Knockdown of A20 regulating IKK activity in rats can increase NF- abnormal B signaling pathway no control, ultimately due to multiple organ failure and inflammatory response to tissue injury and death. As a regulatory protein, A20 deletion can lead to abnormal TLR signal transduction and increase the body's inflammatory reaction degree. The experimental study of interleukin 17 (IL-17) stimulation Inflammatory cytokines of rat spinal cord astrocytes, and the change of A20 protein; expression of MI R-136-5p gene silencing in rat astrocytes, the change of A20 protein in rat astrocytes inflammatory reaction induced by IL-17. To provide a theoretical basis and new method for clinical treatment of acute spinal cord injury. Objective: To investigate the inhibition of inflammation A20 the yield of protein induced changes before and after R-136-5p was inhibited in MI rat astrocyte inflammatory response in inflammatory factor IL-17. Methods: (1) isolated and cultured rat astrocytes; (2) using different concentration of IL-17 (10,20,50100200ng/ml) on rat astrocytes stimulated 12h (3;) using the optimal concentration of that in the previous step, stimulation at different time points (3H, 6h, 12h, 24h and 48h) of the rat astrocytes; (4) by Q PCR and ELISA were detected after IL-17 stimulation Astrocyte inflammatory factors (IL-6, MIP-2, MCP-1, TNF- alpha, MCP-5) (5). The changes of astrocytes were the optimal concentration, the optimum time of stimulation, and by Q PCR and WB A20 m method is used to measure the RNA and protein expression level. (6) the expression of sink silent rat astrocyte Mi R-136-5p gene; and then stimulated by IL-17 and Q PCR R-136-5p determination of MI gene expression level, WB was used to detect the expression of rat astrocytes A20 protein. Results: the experimental group of rat astrocytes after stimulated by IL-17, A20 protein expression in 6h was significantly reduced (P0.05), to 12h at peak, but no significant difference compared with 6h changes; under the induction of IL-17, and the control group (blank) and negative transfection group (control), increased expression of MI R-136-5p gene silencing group in the amount of A20 significantly. Conclusion: rat astrocytes A2 Expression of inflammatory cytokines in 0 had negative correlation in peak expression, A20 protein play a regulatory role in the negative regulation of inflammatory inflammation in rat astrocytes. After silencing the expression of astrocytes of rat mi R-136-5p again IL-17 stimulation, R-136-5p and A20 Mi showed that there is a negative role. Therefore, IL-17 induced inflammation in rat astrocytes, silencing of MI R-136-5p by up regulating the expression of zinc finger protein A20 play an anti-inflammatory effect.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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