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  本文關鍵詞： miR-136-5p 急性脊髓損傷 炎癥 A20　出處：《廣西醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：星形膠質(zhì)細胞在神經(jīng)系統(tǒng)中廣泛存在,并與神經(jīng)元以及其他膠質(zhì)細胞共同構成中樞神經(jīng)系統(tǒng)。星形膠質(zhì)細胞除填充以及營養(yǎng)神經(jīng)元的作用外,亦在神經(jīng)系統(tǒng)的發(fā)育以及修復中也占有一席之地。急性脊髓損傷(ASCI)發(fā)生后,除直接暴力所帶來的原發(fā)性損傷外,多數(shù)患者在數(shù)天內(nèi)將發(fā)生繼發(fā)性脊髓損傷(SSCI)。原發(fā)性損傷是外力直接作用所帶來的直接性物理傷害,同其他意外傷害一樣無直接有效的方法可以針對損傷進行治療;繼發(fā)性脊髓損傷是直接性損傷發(fā)生后,一段時間之內(nèi)因為身體免疫系統(tǒng)對損傷組織激活,所導致的以細胞炎性反應損傷,并最終導致直接損傷部位的加重以及范圍擴大�，F(xiàn)有的資料表明,急性脊髓損傷中,最終要的病理生理變化是因為炎性因子所導致的細胞自我凋亡,這也是急性脊髓損傷中組織損傷較原發(fā)性損傷擴大并加重的重要機制。趨化因子(chemotactic factor)是一類具有促炎和遷移作用的小分子物質(zhì)。通常在炎癥反應中作為免疫細胞的化學誘導物,引導免疫細胞到達炎性反應區(qū)域發(fā)揮吞噬作用。趨化因子及其受體在中樞神經(jīng)系統(tǒng)中廣泛表達,對于干細胞的遷移、軸突的生長和神經(jīng)傳導的調(diào)節(jié)有重要作用。例如SCI后給予XCL-10封閉抗體進行治療可以顯著降低炎癥反應,增強功能的恢復、組織和神經(jīng)元軸突再生。如,有研究發(fā)現(xiàn),外周神經(jīng)損傷后,CXCL-12(又稱MIP-2)可以通過促進中樞神經(jīng)系統(tǒng)髓鞘的生成以及神經(jīng)突的生長,增強SCI后軸突再生和重建。微小RNA(Micro-RNA或mi RNA)是一類小型非編碼RNA分子,可通過堿基互補配對與靶基因的3'非翻譯區(qū)(3'-ntranslated region,3'-UTR)結(jié)合而進而調(diào)控編碼基因的表達。大量研究表明,mi RNAs可廣泛參與腫瘤形成、免疫性疾病等幾乎多種疾病的病理過程。mi R-136為mi RNA家族成員之一,在以往研究中發(fā)現(xiàn),mi R-136在非小細胞癌中較其他組織表達量出現(xiàn)上調(diào),上調(diào)程度與腫瘤的惡性程度及是否容易發(fā)生轉(zhuǎn)移密切相關。另有研究指出mi R-136可通過下調(diào)通道蛋白Smad2和Smad3抑制肺腺癌細胞的轉(zhuǎn)移與侵襲,并通過下調(diào)星形膠質(zhì)細胞抑制膠質(zhì)瘤細胞的凋亡。A20通常也被稱為TNF-α誘導蛋白3(TNFAIP3)或鋅指蛋白3,可以在內(nèi)皮細胞受到TNF-α刺激后產(chǎn)生。A20的保護作用在天然免疫、繼發(fā)性免疫中均表現(xiàn)出明顯效果,部分研究也指出A20具有抑制B細胞腫瘤的發(fā)生。另外,A20是終止激活后的NF-κB關鍵蛋白,并由一些受體如TNF受體、Toll樣受體、核苷酸結(jié)合寡聚化結(jié)構域蛋白2的等調(diào)節(jié)。敲除A20的大鼠體內(nèi)IKK活性會病態(tài)增加使NF-κB信號通路不受控制,最終因多器官炎癥反應衰竭以及組織損傷而死亡。作為一種調(diào)控蛋白,A20缺失也會導致TLR信號傳導的異常并加重機體自身炎癥反應的程度。本實驗研究白介素17(IL-17)刺激大鼠脊髓星形膠質(zhì)細胞產(chǎn)生炎性因子,以及A20蛋白的變化;沉默大鼠星形膠質(zhì)細胞中mi R-136-5p基因的表達,IL-17誘導的大鼠星形膠質(zhì)細胞炎癥反應中A20蛋白變化。為急性脊髓損傷的臨床治療提供理論依據(jù)和新方法。目的:探討A20炎癥抑制蛋白在炎癥因子IL-17誘導大鼠星形膠質(zhì)細胞炎癥反應中于mi R-136-5p被抑制前后的產(chǎn)量變化。方法:(1)體外分離以及培養(yǎng)大鼠星形膠質(zhì)細胞;(2)使用不同濃度的IL-17(10、20、50、100、200ng/ml)對獲得的大鼠星形膠質(zhì)細胞刺激12h;(3)使用上一步中獲知的最適濃度,刺激不同時間點(3h、6h、12h、24h和48h)的大鼠星形膠質(zhì)細胞;(4)通過q PCR以及ELISA的方法分別檢測IL-17刺激之后大鼠星形膠質(zhì)細胞炎癥因子(IL-6、TNF-α、MIP-2、MCP-1、MCP-5)的改變情況。(5)對星形膠質(zhì)細胞進行最適濃度、最適時間的刺激,并通過q PCR以及WB方法分別測量其A20的m RNA以及蛋白表達水平。(6)沉默大鼠星形膠質(zhì)細胞mi R-136-5p的基因表達;然后予IL-17刺激并q PCR測定mi R-136-5p基因水平表達,WB方法檢測大鼠星形膠質(zhì)細胞的A20蛋白表達水平。結(jié)果:實驗組大鼠星形膠質(zhì)細胞受IL-17刺激后,A20蛋白在6h時表達量顯著減少(p0.05),至12h時至峰值,但是與6h相比變化無統(tǒng)計學差異;IL-17誘導下,與空白對照組(blank)以及陰性轉(zhuǎn)染組(control)比較,mi R-136-5p基因沉默組中A20的表達量顯著性的增加。結(jié)論:大鼠星形膠質(zhì)細胞A20的表達量相關炎癥因子變化在峰值的表達量上出現(xiàn)了負相關性,A20炎癥負調(diào)控蛋白在大鼠星形膠質(zhì)細胞炎癥發(fā)揮調(diào)控作用。沉默大鼠星形膠質(zhì)細胞mi R-136-5p的表達之后再次進行IL-17刺激,表明mi R-136-5p與A20之間存在著負性調(diào)控作用。因此,IL-17誘導的大鼠星形膠質(zhì)細胞炎癥中,沉默mi R-136-5p通過上調(diào)鋅指蛋白A20發(fā)揮抗炎作用。
[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.

【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R651.2

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