本文選題：脂聯(lián)素受體　切入點(diǎn)：類風(fēng)濕關(guān)節(jié)炎　出處：《南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版)》2017年09期 　論文類型：期刊論文

【摘要】：目的 :觀察脂聯(lián)素受體1(adiponectin receptor1,AdipoR1)沉默對(duì)脂多糖(lipopolysaccharide,LPS)誘導(dǎo)的人類風(fēng)濕關(guān)節(jié)炎滑膜成纖維細(xì)胞(MH7A)增殖、凋亡的影響。方法:(1)利用免疫熒光、Western blot鑒定并驗(yàn)證shRNA技術(shù)對(duì)構(gòu)建的AdipoR1沉默MH7A(sh-AdipoR1組)細(xì)胞的干擾效率;(2)分別用CCK8試劑盒、流式細(xì)胞儀檢測(cè)LPS(100 ng/m L)對(duì)sh-AdipoR1組及空載病毒對(duì)照(sh-NC組)細(xì)胞增殖及凋亡的影響;(3)利用實(shí)時(shí)定量聚合酶鏈反應(yīng)法(real-time polymerase chain reaction,realtime PCR)檢測(cè)LPS對(duì)sh-AdipoR1及sh-NC組細(xì)胞中凋亡相關(guān)基因BCL-2、BCL-XL、BAX、BAK表達(dá)的影響。同時(shí)設(shè)置無LPS刺激的sh-NC、sh-AdipoR1組作為對(duì)照。結(jié)果:(1)熒光顯微鏡下可見sh-AdipoR1組細(xì)胞熒光蛋白表達(dá)效率趨近于100%,Western blot檢測(cè)結(jié)果顯示:與sh-NC組相比,sh-AdipoR1組AdipoR1蛋白的表達(dá)顯著下降(P0.05),表明AdipoR1沉默的細(xì)胞構(gòu)建成功;(2)無LPS刺激情況下,sh-NC組與sh-AdipoR1組細(xì)胞增殖速率及凋亡細(xì)胞比例無明顯差異;LPS刺激可顯著增加sh-NC組和sh-AdipoR1組細(xì)胞相對(duì)增殖速率及凋亡細(xì)胞比例(P0.05);在LPS作用24、48 h,sh-AdipoR1組細(xì)胞增殖速率均顯著低于sh-NC組(P0.05),而sh-AdipoR1組細(xì)胞凋亡率顯著高于sh-NC組(P0.05);(3)real-time PCR結(jié)果顯示,無LPS刺激情況下,sh-NC組與sh-AdipoR1組抑制凋亡基因BCL-2、BCL-XL與促進(jìn)凋亡基因BAK、BAX的相對(duì)表達(dá)量均無顯著差異(P0.05);LPS刺激可降低抑凋亡基因BCL-2、BCL-XL表達(dá),增加促凋亡基因BAX、BAK表達(dá)(P0.05);在LPS誘導(dǎo)下與sh-NC組相比,sh-AdipoR1組抑制凋亡基因BCL-2、BCL-XL表達(dá)下降,促凋亡基因BAX、BAK表達(dá)顯著升高(P0.05)。結(jié)論:AdipoR1基因的沉默可抑制LPS誘導(dǎo)的MH7A細(xì)胞增殖,促進(jìn)細(xì)胞凋亡,并提示AdipoR1相關(guān)信號(hào)通路可能在類風(fēng)濕關(guān)節(jié)炎發(fā)生發(fā)展中起到重要作用,阻斷該途徑可有效阻斷LPS誘導(dǎo)的MH7A細(xì)胞炎癥反應(yīng)。
[Abstract]:Aim: to observe the effect of adiponectin receptor 1 adipoR1 (AdipoR1) silencing on the proliferation of human rheumatoid arthritis synovial fibroblasts (MH7A) induced by lipopolysaccharide (LPS). Methods the interference efficiency of shRNA on AdipoR1 silenced MH7A(sh-AdipoR1 cells was identified and verified by Western blot. Methods CCK8 kit was used to identify and verify the interference efficiency of shRNA on AdipoR1 silencing MH7A(sh-AdipoR1 cells. Effect of LPS(100 ng/m L) on cell proliferation and apoptosis in sh-AdipoR1 group and non-loaded virus control sh-NC group by flow cytometry) real-time polymerase chain reactionation realtime PCR was used to detect the apoptosis related gene BCL-2 and BCL-XLBAXBAK in sh-AdipoR1 and sh-NC cells by real-time polymerase chain reactiontime PCR. At the same time, the expression efficiency of fluorescent protein in sh-AdipoR1 group was closer than that in sh-NC group. The results showed that compared with sh-NC group, the expression of AdipoR1 protein in sh-NCncncdipoR1 group was higher than that in sh-NC group. There was no significant difference in cell proliferation rate and ratio of apoptotic cells between sh-AdipoR1 group and AdipoR1 group without LPS stimulation. LPS-induced cell proliferation rate increased significantly in sh-NC group and sh-AdipoR1 group. The percentage of apoptotic cells in the LPS group was significantly lower than that in the sh-NC group (P 0.05), and the apoptosis rate in the sh-AdipoR1 group was significantly higher than that in the sh-NC group. The results of real-time PCR showed that the cell proliferation rate in the LPS group was significantly lower than that in the sh-NC group, and that in the sh-AdipoR1 group was significantly higher than that in the sh-NC group, and that in the sh-AdipoR1 group was significantly higher than that in the sh-NC group. There was no significant difference in the relative expression of BCL-2nBCL-XL between the control group and the sh-AdipoR1 group without LPS stimulation. P0.05LPs could decrease the expression of BCL-2nBCL-XL. The expression of BCL-XL, BCL-2nBCL-XL and the expression of BCL-XL in BCL-2nBCL-XL in LPS group were significantly decreased compared with those in sh-NC group. Conclusion the silencing of Bax-AdipoR1 gene can inhibit the proliferation of MH7A cells induced by LPS and promote cell apoptosis. It is suggested that AdipoR1 related signaling pathway may play an important role in the pathogenesis and development of rheumatoid arthritis. Blocking this pathway can effectively block the inflammatory response of MH7A cells induced by LPS.
【作者單位】： 南京醫(yī)科大學(xué)第一附屬醫(yī)院風(fēng)濕免疫科;
【基金】：國(guó)家自然科學(xué)基金(81671615) 重點(diǎn)病種規(guī)范化診療研究(BL20130134) 江蘇省科技廳基礎(chǔ)研究計(jì)劃(BK2012875)
【分類號(hào)】：R593.22

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