發(fā)布時(shí)間：2018-03-11 16:24

  本文選題：自噬　切入點(diǎn)：類(lèi)風(fēng)濕關(guān)節(jié)炎　出處：《川北醫(yī)學(xué)院》2015年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的： 1.初步探討體外培養(yǎng)的類(lèi)風(fēng)濕關(guān)節(jié)炎滑膜成纖維細(xì)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(jī)制。2.RASFs在不同濃度的甲氨蝶呤(Methotrexate,MTX)作用24小時(shí)后,分別在蛋白和基因水平測(cè)定自噬標(biāo)記物微管相關(guān)蛋白1輕鏈3(Microtuble-associated Protein1 light chain3,LC3)和P62的表達(dá)水平,初步探討MTX對(duì)RASFs中自噬水平的影響。方法:分別收集膝關(guān)節(jié)置換的8例類(lèi)風(fēng)濕關(guān)節(jié)炎(Rheumatoid Arthritis,RA)患者和5例骨關(guān)節(jié)炎(Osteoarthritis,OA)患者的滑膜組織,用酶消化法消化后進(jìn)行培養(yǎng),加入不同濃度的MTX作用24小時(shí)后,利用蛋白質(zhì)印記法(Western-blotting,WB)檢測(cè)LC3蛋白、P62蛋白的表達(dá)水平;實(shí)時(shí)定量反轉(zhuǎn)錄-聚合酶連反應(yīng)(RT-q PCR)方法檢測(cè)LC3mRNA、P62m RNA的表達(dá)水平;利用透射電鏡檢測(cè)自噬體。結(jié)果： 1.WB結(jié)果:LC3蛋白、P62蛋白在RASFs和OASFs中均有表達(dá),RASFs空白組與OASFs空白組相比,LC3蛋白表達(dá)增加、P62蛋白表達(dá)下降。在RASFs組,隨著藥物濃度的增加LC3蛋白表達(dá)下降、P62蛋白表達(dá)增加。2.RT-q PCR結(jié)果:LC3m RNA、P62m RNA在RASFs和OASFs中均有表達(dá)。RASFs空白組與OASFs空白組相比,LC3m RNA表達(dá)增加、P62m RNA表達(dá)下降。RASFs中藥物處理各組與空白組相比無(wú)明顯差異無(wú)統(tǒng)計(jì)學(xué)意義。3.透射電鏡結(jié)果:在RASFs和OASFs中均可以檢測(cè)到自噬體的形成。結(jié)論： 1.RASFs中自噬水平升高;2.RASFs饑餓24小時(shí)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(qiáng)。利用自噬抑制劑治療滑膜增生有望成為可能。4.MTX對(duì)RASFs中LC3m RNA、P62m RNA的表達(dá)無(wú)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(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R593.22

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本文編號(hào)：1598953