低氧诱导microRNA-146a促进软骨细胞自噬的作用机制研究

发布时间：2018-03-16 17:27

本文选题：骨性关节炎　切入点：软骨细胞　出处：《第二军医大学》2016年硕士论文　论文类型：学位论文

【摘要】：研究背景与目的目前关于骨关节炎(Osteoarthritis,OA)的病因研究已经有很多,例如促炎细胞因子的作用,软骨降解反应,miR-146a的调控作用,低氧刺激和骨关节炎中软骨细胞的自噬作用,但是却没有关于软骨细胞中miR-146a和自噬之间关系的报道,特别是低氧环境下的关系。本实验旨在明确软骨在低氧条件下miR-146a和软骨细胞自噬之间的作用关系。方法软骨细胞培养在不同程度氧含量的条件下,通过定量聚合酶链反应(Quantitative Polymerase Chain Reaction,QPCR)和免疫印迹试验(Western blot)检测主要细胞因子HIF-1α,HIF-2α,MiR-146a,Bcl-2和自噬标记基因ULK-1,ATG-5的表达。自噬标记基因LC-3则通过免疫荧光法检测。通过基因水平的过表达和下调操作,结合定量聚合酶链反应和免疫印迹试验等方法检测mRNA和蛋白,证实miR-146a与Bcl-2的相互作用。结果低氧条件能够维持软骨细胞表型,促进细胞自噬,并通过HIF-1α而不是HIF-2α促进miR-146a的表达,但是miR-146a对HIF-1α并无反作用。低氧诱导细胞自噬需要HIF-1α,miR-146a和Bcl-2的介入。低氧诱导HIF-1α的表达,HIF-1α促进miR-146a的表达,而miR-146a却显著阻止自噬抑制剂Bcl-2的表达,从而促进细胞自噬。但是Bcl-2对HIF-1α和miR-146a的表达则均无影响。HIF-1α与miR-146a的缺失或者Bcl-2的过表达都会抑制低氧诱导的细胞自噬。结论HIF-1α,MiR-146a和Bcl-2在低氧诱导软骨细胞自噬中都产生关键作用。低氧条件下,HIF-1α和MiR-146a通过抑制Bcl-2的表达而促进软骨细胞自噬。我们推断,miR-146a可能将成为阻止骨性关节炎软骨退变的新治疗靶点。
[Abstract]:Background and objective there have been many studies on the etiology of Osteoarthritis Osteoarthritis (Osteoarthritis), such as the role of pro-inflammatory cytokines, the regulatory effect of cartilage degradation response (miR-146a), the role of hypoxia-stimulated chondrocytes and autophagy of chondrocytes in osteoarthritis. But there was no report of the relationship between miR-146a and autophagy in chondrocytes. The purpose of this study was to clarify the relationship between miR-146a and autophagy of chondrocytes under hypoxic conditions. Methods chondrocytes were cultured under different levels of oxygen content. The expression of major cytokines (HIF-1 伪, HIF-2 伪, MiR-146a, Bcl-2 and ULK-1 / ATG-5) was detected by quantitative Polymerase Chain reaction (Polymerase) and Western blot assay (Western blot). The autophagic marker gene (LC-3) was detected by immunofluorescence assay. Expression and down-regulation operations, The interaction between miR-146a and Bcl-2 was confirmed by quantitative polymerase chain reaction and Western blotting. Results hypoxia could maintain the phenotype of chondrocytes, promote autophagy, and promote the expression of miR-146a through HIF-1 伪 instead of HIF-2 伪. However, miR-146a had no reaction to HIF-1 伪. Hypoxia induced autophagy required the intervention of HIF-1 伪 miR-146a and Bcl-2. Hypoxia induced HIF-1 伪 expression increased miR-146a expression, while miR-146a significantly inhibited the expression of autophagy inhibitor Bcl-2. But Bcl-2 had no effect on the expression of HIF-1 伪 and miR-146a. HIF-1 伪 and miR-146a deletion or over-expression of Bcl-2 could inhibit hypoxia induced autophagy. Conclusion HIF-1 伪 MiR-146a and Bcl-2 can both produce in hypoxia-induced chondrocyte autophagy. HIF-1 伪 and MiR-146a promote chondrocyte autophagy by inhibiting the expression of Bcl-2 under hypoxia. We infer that miR-146a may be a new therapeutic target to prevent cartilage degeneration in osteoarthritis.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R684.3

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