【摘要】：【目的】肘管綜合征(Cubital tunnel syndrome,CuTS),也稱遲發(fā)性尺神經(jīng)炎,是第二常見的周圍神經(jīng)卡壓病變。肘部的創(chuàng)傷及慢性勞損可以導致弓狀韌帶出現(xiàn)肥厚增生,引起尺神經(jīng)卡壓磨損,這是CuTS最常見的病因。在此本課題組選用微陣列芯片檢測弓狀韌帶中差異表達的microRNAs(miRNAs)并行生物信息學分析,進一步對相關通路進行驗證,期望為CuTS的發(fā)病機制和病因?qū)W研究提供新的思路。【方法】1.3例弓狀韌帶標本及3例對照屈肌總腱旁腱性組織標本均收集自2014年8月至2014年11月期間于天津醫(yī)科大學總醫(yī)院行肘部尺神經(jīng)松解前移手術患者。常規(guī)提取總RNA并行微陣列芯片分析,qRT-PCR用于驗證部分miRNAs表達一致情況。2.miRWalk2.0軟件用于預測靶基因。Cytoscape3.0軟件制作miRNAs與相應靶基因關系網(wǎng)絡圖。DAVID6.7軟件、Bioconductor基因注釋軟件用于靶基因的GO功能富集分析和KEGG通路富集分析。通過Cluster3.0軟件對miRNAs及通路行聚類分析。3.構(gòu)建人皮膚成纖維細胞系(Human skin fibroblast cell lines,HSF),并轉(zhuǎn)染miR-146b-5p過表達(miR-146b-5p mimic)及反義干擾載體(anti-miR-146b-5p),通過qRT-PCR及Western-Blot技術檢測纖維化指標COL1A1、COL3A1、Fibronectin(FN)、SMAD4及TGFB1,從而明確miR-146b-5p影響纖維化是否與TGF-β/SMAD4通路有關�！窘Y(jié)果】在弓狀韌帶組織中差異表達的miRNAs共有70個(P0.05),其中上調(diào)3個(hsa-miR-422a、hsa-miR-7855-5p、hsa-miR-1343-3p);下調(diào)67個(hsa-miR-196a-5p、hsa-miR-146b-5p、hsa-miR-297、hsa-miR-21-3p、hsa-miR-595、hsa-miR-663b、hsa-miR-615-5p、hsa-miR-185-3p等)。qRT-PCR驗證結(jié)果表明hsa-miR-1343-3p、hsa-miR-146b-5p和hsa-miR-21-3p表達與芯片結(jié)果一致。通過miRWalk2.0軟件共發(fā)現(xiàn)1804個預測靶基因。對靶基因分析后發(fā)現(xiàn)大部分靶基因主要受7個miRNAs(hsa-miR-146b-5p,hsa-miR-21-3p,hsa-miR-185-3p,hsa-miR-615-5p,hsa-miR-659-3p,hsa-miR-663a和hsa-miR-760)的調(diào)控。通過Cluster3.0軟件對miRNAs行聚類分析,結(jié)果可見hsa-miR-146b-5p與hsa-miR-196a-5p、hsa-miR-659-3p功能相聚類,hsa-miR-21-3p與hsa-miR-297功能相聚類。對全部靶基因分析發(fā)現(xiàn),WNT2、TGFB1、FGF1等均為差異miRNAs的高頻靶基因并參與體內(nèi)蛋白質(zhì)代謝過程,細胞生長調(diào)節(jié),細胞周期過程,細胞分裂,細胞代謝過程,信號傳導等生物過程。KEGG通路分析示靶基因主要集中在粘著連接、粘附斑、軸突導向、賴氨酸降解、其他多糖降解、細胞粘附分子(Cell adhesion molecules,CAMs)、絲裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)信號通路、逆行內(nèi)源性大麻素信號、粘多糖的生物合成-硫酸乙酰肝素/肝素和神經(jīng)營養(yǎng)因子的信號轉(zhuǎn)導通路及其他8條通路。通過Cluster3.0軟件對全部通路行聚類分析,發(fā)現(xiàn)粘附斑與ErbB信號通路功能相聚類,粘著連接與CAMs功能相聚。MAPK、粘附斑信號通路和預測靶基因關系最緊密。qRT-PCR及Western-Blot驗證發(fā)現(xiàn)miR-146b-5p mimic組中COL1A1、COL3A1、FN、SMAD4及TGFB1各纖維化指標均下調(diào),anti-miR-146b-5p組中各纖維化指標均上調(diào)�！窘Y(jié)論】通過分析CuTS患者中退變增生性弓狀韌帶與對照腱性組織的miRNAs表達,發(fā)現(xiàn)了多個差異表達的miRNAs,并獲得了潛在的靶基因及信號通路,為初步研究CuTS中弓狀韌帶增生肥厚的發(fā)病機制提供了線索。
[Abstract]:[Objective] Cubital tunnel syndrome (CuTS), also known as delayed ulnar neuritis, is the second most common peripheral nerve entrapment lesion. CuTS is the most common cause of ulnar nerve entrapment. CuTS is caused by the hypertrophy of the arcuate ligament and ulnar nerve entrapment wear caused by elbow trauma and chronic strain. Detection of differentially expressed microRNAs (microRNAs) in the arcuate ligament and bioinformatics analysis were performed to further validate the related pathways and provide new ideas for the pathogenesis and etiology of CuTS. [Methods] 1.3 specimens of the arcuate ligament and 3 specimens of the paratendinous tissue of the common flexor tendon were collected from August 2014 to 2011. Patients undergoing ulnar nerve release and anterior displacement in Tianjin Medical University General Hospital in January. Total RNA was routinely extracted and analyzed by microarray chip. QRT-PCR was used to verify the consistent expression of some microRNAs. 2. MicroWalk 2.0 software was used to predict target genes. Cytoscape 3.0 software was used to make a network diagram of the relationship between microRNAs and the corresponding target genes. DAVID 6.7 software, B. Ioconductor gene annotation software was used for GO function enrichment analysis and KEGG pathway enrichment analysis of target genes.Cluster 3.0 software was used for clustering analysis of microRNAs and pathways.3. Human skin fibroblast cell lines (HSF) were constructed and transfected with overexpression of microRNAs-146b-5p and antisense interference vector (anti-m-m). IR-146b-5p, COL1A1, COL3A1, Fibronectin (FN), SMAD4 and TGFB1 were detected by qRT-PCR and Western-Blot techniques to determine whether the effect of microwave-146b-5p on fibrosis was related to the TGF-beta/SMAD4 pathway. [Results] There were 70 differentially expressed microRNAs in arcuate ligament tissues (P 0.05). Three of them were up-regulated (hsa-microwave-422a, hsa-microwave-78). 55-5p, hsa-microRNA-1343-3p; 67 down-regulated (hsa-microRNA-196a-196a-5p, hsa-microRNA-196a-196a-5p, hsa-microRNA-146b-5p, hsa-microRNA-297, hsa-microRNA-297, hsa-microRNA-microRNA-21-21-3p, hsa-microRNA-595, hsa-microRNA-595, hsa-microRNA-microRNA-595, hsa-microRNA-microRNA-66-663b, hsa-microRNA-615-5p, hsa-microRNA-microRNA-185-3p and so on). qRT-PCR validation results showed that hsa-microRNA-microRNA-microRNA-1343-The expression of -3p was consistent with the result of microarray. A total of 1804 predictive targets were found by using the microarray Walk 2.0 software. Most of the target genes were mainly regulated by seven microRNAs (hsa-microRNA-146b-5p, hsa-microRNA-146b-5p, hsa-microRNA-21-3p, hsa-microRNA-185-3p, hsa-microRNA-185-3p, hsa-microRNA-615-5p, hsa-microRNA-659-3p, hsa-microRNA-microRNA-663a and hsa-microRNA-663a-760). Cluster3.0 software was used to cluster analysis of microRNA. The results showed that hsa-microRNA-microRNA-146b-146b-5p and hsa-microRNA-5p and hsa-microRNA-1965-5p, hsa-196a-196a-196a-659-3p function The analysis of all target genes showed that WNT2, TGFB1 and FGF1 were high-frequency target genes of differentially expressed microRNAs and were involved in biological processes such as protein metabolism, cell growth regulation, cell cycle process, cell division, cell metabolism and signal transduction. Because of the main focus on adhesion junction, adhesion plaque, axon-directed, lysine degradation, other polysaccharide degradation, cell adhesion molecules (CAMs), mitogen-activated protein kinase (MAPK) signaling pathway, retrograde endocannabinoid signal, mucopolysaccharide biosynthesis - heparan sulfate / heparin Cluster analysis of all the pathways by Cluster 3.0 software showed that adhesion plaques were clustered with ErbB signaling pathway function, adhesion junctions were clustered with CAMs function. MAPK, adhesion plaque signaling pathway and predictive target genes were most closely related. QRT-PCR and Western-Blot validation showed that microRNA146b was found. COL1A1, COL3A1, FN, SMAD4 and TGFB1 were down-regulated in the -5p mimic group, and all fibrosis indexes were up-regulated in the anti-microRNA-146b-5p group. [Conclusion] By analyzing the expression of microRNAs in the degenerative proliferative arcuate ligament and the control tendinous tissue in CuTS patients, multiple differentially expressed microRNAs were found and potential target genes and messages were obtained. The pathway provides clues for the preliminary study of the pathogenesis of arcuate ligament hyperplasia and hypertrophy in CuTS.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R688

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