自噬在类风湿关节炎滑膜成纤维细胞中的作用及甲氨蝶呤对自噬的影响

发布时间：2018-03-11 16:24

  本文选题：自噬　切入点：类风湿关节炎　出处：《川北医学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的： 1.初步探讨体外培养的类风湿关节炎滑膜成纤维细胞(Rheumatoid Arthritis Synovial Fibroblasts,RASFs)中是否存在自噬现象及自噬在RA中的发病机制。2.RASFs在不同浓度的甲氨蝶呤(Methotrexate,MTX)作用24小时后,分别在蛋白和基因水平测定自噬标记物微管相关蛋白1轻链3(Microtuble-associated Protein1 light chain3,LC3)和P62的表达水平,初步探讨MTX对RASFs中自噬水平的影响。方法:分别收集膝关节置换的8例类风湿关节炎(Rheumatoid Arthritis,RA)患者和5例骨关节炎(Osteoarthritis,OA)患者的滑膜组织,用酶消化法消化后进行培养,加入不同浓度的MTX作用24小时后,利用蛋白质印记法(Western-blotting,WB)检测LC3蛋白、P62蛋白的表达水平;实时定量反转录-聚合酶连反应(RT-q PCR)方法检测LC3mRNA、P62m RNA的表达水平;利用透射电镜检测自噬体。结果： 1.WB结果:LC3蛋白、P62蛋白在RASFs和OASFs中均有表达,RASFs空白组与OASFs空白组相比,LC3蛋白表达增加、P62蛋白表达下降。在RASFs组,随着药物浓度的增加LC3蛋白表达下降、P62蛋白表达增加。2.RT-q PCR结果:LC3m RNA、P62m RNA在RASFs和OASFs中均有表达。RASFs空白组与OASFs空白组相比,LC3m RNA表达增加、P62m RNA表达下降。RASFs中药物处理各组与空白组相比无明显差异无统计学意义。3.透射电镜结果:在RASFs和OASFs中均可以检测到自噬体的形成。结论： 1.RASFs中自噬水平升高;2.RASFs饥饿24小时后自噬水平明显下降;3.MTX能抑制RASFs自噬,且随着药物浓度增大,抑制作用增强。利用自噬抑制剂治疗滑膜增生有望成为可能。4.MTX对RASFs中LC3m RNA、P62m RNA的表达无影响。
[Abstract]:Objective: 1. To investigate whether there is autophagy in Rheumatoid Arthritis Synovial FibroblastsFs and the pathogenesis of autophagy in RA in vitro. 2. After 24 hours of exposure to different concentrations of methotrexate MTX, RASFs were used in the treatment of rheumatoid arthritis synovial fibroblasts. The expression levels of microtubule-associated protein 1 light chain 3tuble-associated Protein1 light chain3LC3 and P62 were measured at the protein and gene levels, respectively. Methods: synovial tissues of 8 patients with rheumatoid arthritis (RA) and 5 patients with osteoarthritis (Osteoarthritis) were collected and cultured by enzyme digestion. After 24 hours of exposure to different concentrations of MTX, protein imprinting method was used to detect the expression level of LC3 protein P62, and real-time quantitative reverse transcription-polymerase chain reaction (RT-q PCR) was used to detect the expression level of LC3mRNA-P62m RNA. Results: 1. Wideband results: in RASFs and OASFs, the expression of P62 protein in RASFs and OASFs increased, and the expression of P62 protein decreased in RASFs group, compared with that in OASFs blank group, and the expression of P62 protein was decreased in RASFs group, and the expression of P62 protein in OASFs group was significantly decreased compared with that in OASFs blank group, and the expression of P62 protein was decreased in RASFs group. The expression of LC3 protein decreased with the increase of drug concentration. 2. RT-Q PCR results the expression of P62m RNA in RASFs and OASFs was increased in RASFs and OASFs. Compared with the control group of OASFs, the expression of RNA in RASFs increased. The expression of P62m RNA decreased in the drug treatment group. The results of transmission electron microscope showed that autophagy formation could be detected in both RASFs and OASFs. Conclusion: 1.The level of autophagy in RASFs increased 2.After starvation for 24 hours, the level of autophagy decreased significantly. 3. MTX could inhibit RASFs autophagy. With the increase of drug concentration, the inhibitory effect was enhanced. It may be possible to use autophagy inhibitor to treat synovial hyperplasia. 4. MTX has no effect on the expression of LC3m RNA-P62m RNA in RASFs.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.22

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