本文選題：類風(fēng)濕關(guān)節(jié)炎　切入點(diǎn)：樹突狀細(xì)胞　出處：《廣州中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：目的:通過體外實(shí)驗(yàn),研究斷藤益母湯對類風(fēng)濕關(guān)節(jié)炎(rheumatoid arthritis,RA)患者外周血樹突狀細(xì)胞(dendriticcell,DC)遷移功能相關(guān)的趨化因子和受體表達(dá)的影響,從DC遷移功能的角度來探討斷藤益母湯的免疫抑制和治療RA的機(jī)制。方法:密度梯度離心法分離RA患者外周血單個(gè)核細(xì)胞(PBMCs),采用貼壁法分離出單核細(xì)胞,并以粒細(xì)胞巨噬細(xì)胞集落刺激因子(GM-CSF)及白介素-4(IL-4)刺激7天以使單核細(xì)胞分化成樹突狀細(xì)胞,分別加入甲氨蝶呤(陽性對照)、斷藤益母湯凍干粉(試驗(yàn)組)進(jìn)行干預(yù),隨后以酶聯(lián)免疫吸附測定(ELISA)測定上清液中細(xì)胞因子CXCL9和CXCL10的含量,并收集細(xì)胞,利用流式細(xì)胞技術(shù)檢測DC表面CCR5和CCR7的表達(dá)水平,并與空白組進(jìn)行對比。結(jié)果:1.在GM-CSF和IL-4的共同刺激下,外周血單核細(xì)胞成功分化成為DC,在光鏡下可觀察到DC的典型樹突狀形態(tài)。2.根據(jù)CCK8的細(xì)胞毒理-增殖實(shí)驗(yàn)結(jié)果,比較相同時(shí)間點(diǎn)下,不同斷藤益母湯濃度組與正常組的A值,得出斷藤益母湯凍干粉的最佳給藥質(zhì)量體積比為l00mg/ml。3.經(jīng)ELISA法檢測后,結(jié)果顯示,與空白組對比,經(jīng)斷藤益母湯凍干粉及甲氨蝶呤干預(yù)后DC培養(yǎng)的上清液中CXCL9、CXCL10濃度均明顯下降(P0.05),差異具有統(tǒng)計(jì)學(xué)意義;而斷藤益母湯凍干粉干預(yù)組與甲氨蝶呤組對比,CXCL9、CXCL10濃度下降不明顯(P0.05),差異沒有統(tǒng)計(jì)學(xué)意義。4.流式細(xì)胞結(jié)果顯示,經(jīng)斷藤益母湯凍干粉及甲氨蝶呤干預(yù)后的DC表面CCR5和CCR7的表達(dá)均明顯下調(diào)(P0.05),差異具有統(tǒng)計(jì)學(xué)意義;而斷藤益母湯凍干粉干預(yù)組與甲氨蝶呤組對比,CCR5和CCR7的表達(dá)略有下調(diào)(P0.05),但差異沒有統(tǒng)計(jì)學(xué)意義。結(jié)論:斷藤益母湯可以下調(diào)RA患者外周血DC表面CCR5和CCR7的表達(dá),減少趨化因子CXCL9和CXCL10的分泌,從而通過抑制DC往次級淋巴器官的遷移,減少其與T細(xì)胞的特異性結(jié)合,從而起到治療RA的作用。
[Abstract]:Objective: to study the effect of Shuteng Yimu decoction on the expression of chemokines and receptors related to the migration function of peripheral blood dendritic cells (DC) in patients with rheumatoid arthritis (RA).From the point of view of DC migration function, the mechanism of immunosuppression and treatment of RA was studied.Methods: peripheral blood mononuclear cells from patients with RA were isolated by density gradient centrifugation. Monocytes were isolated by adherent method and stimulated with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4IL-4 (IL-4) for 7 days to differentiate monocytes into dendritic cells.Methotrexate (positive control group, Shutentenyimu decoction freeze-dried powder) was added to the experiment group, then the levels of cytokines CXCL9 and CXCL10 in the supernatant were measured by enzyme-linked immunosorbent assay (Elisa), and the cells were collected.Flow cytometry was used to detect the expression of CCR5 and CCR7 on DC surface and compared with the blank group.The result is 1: 1.Under the co-stimulation of GM-CSF and IL-4, peripheral blood mononuclear cells were successfully differentiated into DC.The typical dendritic morphology of DC was observed under light microscope.According to the results of cell toxicology and proliferation test of CCK8, the A value of different concentration groups and normal group were compared at the same time point, and the optimum volume ratio of the lyophilized powder of Shuteng Yimu decoction was 1 00mg / ml 路ml. 3.The results of ELISA test showed that the concentration of CXCL9 and CXCL10 in the supernatant of DC cultured after the intervention of Shuteng Yimu decoction and methotrexate was significantly lower than that of the blank group, and the difference was statistically significant.The concentration of CXCL9 and CXCL10 in the intervention group and methotrexate group were not significantly decreased (P 0.05), and there was no significant difference between them.The results of flow cytometry showed that the expression of CCR5 and CCR7 on the DC surface was significantly down-regulated after the intervention of Shutenyimu decoction freeze-dried powder and methotrexate, and the difference was statistically significant.Compared with methotrexate group, the expression of CCR5 and CCR7 was slightly down-regulated, but there was no significant difference in the expression of CCR5 and CCR7 between the intervention group and the methotrexate group.Conclusion: TYD can down-regulate the expression of CCR5 and CCR7 on peripheral blood DC of RA patients, reduce the secretion of chemokine CXCL9 and CXCL10, and reduce the specific binding of DC to T cells by inhibiting the migration of DC to secondary lymphoid organs.So as to play a role in the treatment of RA.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R259

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