本文選題：自噬　切入點：類風濕關節(jié)炎　出處：《川北醫(yī)學院》2015年碩士論文　論文類型：學位論文

【摘要】：目的： 1.初步探討體外培養(yǎng)的類風濕關節(jié)炎滑膜成纖維細胞(Rheumatoid Arthritis Synovial Fibroblasts,RASFs)中是否存在自噬現(xiàn)象及自噬在RA中的發(fā)病機制。2.RASFs在不同濃度的甲氨蝶呤(Methotrexate,MTX)作用24小時后,分別在蛋白和基因水平測定自噬標記物微管相關蛋白1輕鏈3(Microtuble-associated Protein1 light chain3,LC3)和P62的表達水平,初步探討MTX對RASFs中自噬水平的影響。方法:分別收集膝關節(jié)置換的8例類風濕關節(jié)炎(Rheumatoid Arthritis,RA)患者和5例骨關節(jié)炎(Osteoarthritis,OA)患者的滑膜組織,用酶消化法消化后進行培養(yǎng),加入不同濃度的MTX作用24小時后,利用蛋白質印記法(Western-blotting,WB)檢測LC3蛋白、P62蛋白的表達水平;實時定量反轉錄-聚合酶連反應(RT-q PCR)方法檢測LC3mRNA、P62m RNA的表達水平;利用透射電鏡檢測自噬體。結果： 1.WB結果:LC3蛋白、P62蛋白在RASFs和OASFs中均有表達,RASFs空白組與OASFs空白組相比,LC3蛋白表達增加、P62蛋白表達下降。在RASFs組,隨著藥物濃度的增加LC3蛋白表達下降、P62蛋白表達增加。2.RT-q PCR結果:LC3m RNA、P62m RNA在RASFs和OASFs中均有表達。RASFs空白組與OASFs空白組相比,LC3m RNA表達增加、P62m RNA表達下降。RASFs中藥物處理各組與空白組相比無明顯差異無統(tǒng)計學意義。3.透射電鏡結果:在RASFs和OASFs中均可以檢測到自噬體的形成。結論： 1.RASFs中自噬水平升高;2.RASFs饑餓24小時后自噬水平明顯下降;3.MTX能抑制RASFs自噬,且隨著藥物濃度增大,抑制作用增強。利用自噬抑制劑治療滑膜增生有望成為可能。4.MTX對RASFs中LC3m RNA、P62m RNA的表達無影響。
[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.
【學位授予單位】：川北醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R593.22

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本文編號：1598953