本文關(guān)鍵詞： 類(lèi)風(fēng)濕關(guān)節(jié)炎 骨破壞 外周血單個(gè)核細(xì)胞 破骨細(xì)胞 姜黃素 核因子κB受體活化因子　出處：《安徽中醫(yī)藥大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:掌握外周血單個(gè)核細(xì)胞(PBMCs)體外誘導(dǎo)培養(yǎng)破骨細(xì)胞的方法,以此研究類(lèi)風(fēng)濕關(guān)節(jié)炎(RA)患者外周血破骨細(xì)胞前體細(xì)胞(OPC)的分化能力;并進(jìn)一步研究姜黃素對(duì)RA患者外周血破骨細(xì)胞分化成熟及核因子κB受體活化因子(RANK)表達(dá)的影響;探討破骨細(xì)胞在RA骨質(zhì)破壞中的作用及姜黃素防治RA可能的分子機(jī)制,為中藥單體姜黃素治療RA提供新的實(shí)驗(yàn)依據(jù)。方法:(1)①將12例RA患者和10名健康志愿者分為RA組和健康對(duì)照組,分別抽取兩組人員的外周血,分離PBMCs,采用細(xì)胞因子RANKL(100 nmol/L)和M-CSF(50 nmol/L)體外誘導(dǎo)培養(yǎng)破骨細(xì)胞的方法對(duì)細(xì)胞進(jìn)行培養(yǎng);對(duì)培養(yǎng)14天后的細(xì)胞行抗酒石酸酸性磷酸酶(TRAP)染色,觀察其細(xì)胞形態(tài);將TRAP染色陽(yáng)性、胞核≥3個(gè)的細(xì)胞定義為破骨細(xì)胞并計(jì)數(shù);對(duì)培養(yǎng)21天的細(xì)胞進(jìn)行骨吸收實(shí)驗(yàn);將RA組和健康對(duì)照組的破骨細(xì)胞形態(tài)、數(shù)量及骨吸收功能進(jìn)行比較。②對(duì)RA組患者骨質(zhì)破壞的臨床指標(biāo)進(jìn)行評(píng)估,評(píng)估內(nèi)容包括:X線(xiàn)檢查其雙手關(guān)節(jié)行影像學(xué)Sharp評(píng)分,雙能X線(xiàn)骨密度儀測(cè)試其骨密度(BMD)值;并將RA組破骨細(xì)胞數(shù)量與該組骨質(zhì)破壞的臨床指標(biāo)進(jìn)行相關(guān)分析。(2)①采集3例健康志愿者PBMCs,在含不同濃度(0、2.5、5、10、20、40μmol/L)的姜黃素培養(yǎng)液中分別培養(yǎng)24、48和72 h,采用CCK-8法檢測(cè)其細(xì)胞活力。②采集10例RA患者PBMCs,加入RANKL(100 nmol/L)和M-CSF(50 nmol/L)誘導(dǎo)PBMCs向破骨細(xì)胞分化,在細(xì)胞培養(yǎng)過(guò)程中加入不同濃度姜黃素進(jìn)行干預(yù);根據(jù)姜黃素濃度將實(shí)驗(yàn)分為4組,即空白對(duì)照組、姜黃素2.5μmol/L組、姜黃素5μmol/L組和姜黃素10μmol/L組;對(duì)培養(yǎng)14天后的細(xì)胞行TRAP染色,計(jì)數(shù)TRAP染色陽(yáng)性、胞核≥3個(gè)的細(xì)胞(定義為破骨細(xì)胞),比較各組破骨細(xì)胞數(shù)量。③采集5例RA患者PBMCs,加入RANKL(100 nmol/L)和M-CSF(50 nmol/L)體外誘導(dǎo)培養(yǎng),在培養(yǎng)過(guò)程中加入不同濃度姜黃素進(jìn)行干預(yù);實(shí)驗(yàn)分組同上;在細(xì)胞培養(yǎng)10天后,采用RT-PCR法檢測(cè)各組破骨細(xì)胞前體細(xì)胞RANK m RNA的表達(dá);Western-blot法檢測(cè)各組破骨細(xì)胞前體細(xì)胞RANK蛋白的表達(dá)。結(jié)果:(1)①RA組和健康對(duì)照組的破骨細(xì)胞形態(tài)相似,無(wú)明顯區(qū)別;RA組破骨細(xì)胞數(shù)量(129±6.999個(gè)/10個(gè)視野)較健康對(duì)照組(79±3.887個(gè)/10個(gè)視野)顯著升高(P0.05);RA組破骨細(xì)胞骨吸收功能明顯高于健康對(duì)照組。②RA組破骨細(xì)胞數(shù)量與Sharp評(píng)分呈顯著正相關(guān)(r=0.810,P=0.001),與BMD(T值)呈顯著負(fù)相關(guān)(r=-0.685,P=0.014)。(2)①CCK-8法檢測(cè)結(jié)果顯示較低濃度(2.5μmol/L、5μmol/L、10μmol/L)姜黃素對(duì)PBMCs細(xì)胞活力無(wú)明顯影響,較高濃度姜黃素(20μmol/L和40μmol/L)顯著降低其細(xì)胞活力。②細(xì)胞培養(yǎng)14天后,與空白對(duì)照組(126.3±4.1個(gè)/10個(gè)視野)比較,姜黃素2.5μmol/L組(101.8±3.5個(gè)/10個(gè)視野)、姜黃素5μmol/L組(79.9±3.8個(gè)/10個(gè)視野)、姜黃素10μmol/L組(60.6±4.4個(gè)/10個(gè)視野)破骨細(xì)胞的數(shù)量均明顯減少,且具有濃度依賴(lài)性(P0.05)。③RT-PCR結(jié)果顯示,與空白對(duì)照組(1.44±0.16)比較,姜黃素2.5μmol/L組(1.03±0.12)、姜黃素5μmol/L組(0.48±0.10)和姜黃素10μmol/L組(0.26±0.04)RANK m RNA的表達(dá)均顯著降低,且具有濃度依賴(lài)性(P0.05);Western-blot結(jié)果顯示,與空白對(duì)照組(0.68±0.11)比較,姜黃素2.5μmol/L組(0.46±0.09)、姜黃素5μmol/L組(0.36±0.08)和姜黃素10μmol/L組(0.25±0.07)RANK蛋白的表達(dá)均顯著降低,且具有濃度依賴(lài)性(P0.05)。結(jié)論:(1)RA患者PBMCs向破骨細(xì)胞分化的能力明顯增加,且與RA骨質(zhì)破壞的臨床指標(biāo)密切相關(guān)。這可能是造成RA骨質(zhì)破壞的重要機(jī)制。(2)姜黃素具有體外抑制RA患者PBMCs向破骨細(xì)胞分化的作用,抑制RA患者外周血破骨細(xì)胞前體細(xì)胞RANK m RNA及蛋白表達(dá)的作用,且均呈濃度依賴(lài)性。提示姜黃素可能通過(guò)作用于RANK介導(dǎo)的信號(hào)通路抑制了破骨細(xì)胞的分化、成熟。這可能是姜黃素治療RA骨質(zhì)破壞的重要作用機(jī)制。
[Abstract]:Objective: To investigate the peripheral blood mononuclear cells (PBMCs) induced culture of osteoclasts in vitro, in order to study the rheumatoid arthritis (RA) in peripheral blood of patients with osteoclast precursor cell (OPC) differentiation ability; and further study the effect of curcumin on RA in peripheral blood of patients with broken bone cell differentiation and nuclear receptor factor kappa B activation factor (RANK) on the expression of; to explore the molecular mechanism of osteoclasts in bone destruction in RA and the effect of curcumin in preventing RA possible, provide new experimental basis for traditional Chinese medicine monomer of curcumin in the treatment of RA. Methods: (1) the 12 cases of RA patients and 10 healthy volunteers were divided into RA group and the healthy control group, peripheral blood, extracted from the two groups using cell separation PBMCs, factor RANKL (100 nmol/L) and M-CSF (50 nmol/L) inducing culture methods on osteoclast cells were cultured in vitro; the cells cultured for 14 days for tartrate resistant acid phosphate The enzyme (TRAP) staining, observe the cell morphology; TRAP positive staining, nuclei of not less than 3 of the cells defined as osteoclasts on bone resorption and counting; experiments were performed 21 days of cell culture; the cell morphology of osteoclasts in RA group and healthy control group, and the number of bone resorption were compared. The evaluation of the clinical indicators of RA patients bone destruction, including: X-ray imaging for the joints of the hands of Sharp score, dual energy X-ray absorptiometry to test their bone mineral density (BMD) value; and the RA group was breaking clinical index of bone cell number and destruction of the group of bone were analyzed. (2) the acquisition of 3 healthy volunteers in PBMCs, with different concentrations of curcumin (0,2.5,5,10,20,40 mol/L) in the culture medium were cultured in 24,48 and 72 h, detection of cell viability by CCK-8 assay. The collection of 10 cases of RA patients with PBMCs, RANKL (100 nmol/L) and M-CSF (50 nmol/L) to induce PBMCs The differentiation of osteoclasts, with different concentrations of curcumin in cell culture in the process of intervention; according to the concentration of curcumin were divided into 4 groups, namely control group, curcumin 2.5 mol/L curcumin group, 5 mol/L group and 10 mol/L curcumin group; staining after 14 days of culture cell line TRAP, count of TRAP positive the nucleus, less than 3 of the cells (defined as osteoclasts), compared the number of osteoclasts. The collection of 5 cases of RA patients with PBMCs, RANKL (100 nmol/L) and M-CSF (50 nmol/L) cultured in vitro, with different concentrations of curcumin in the training process of intervention group are the same as above; training; 10 days after the cells were detected the expression of RANK m RNA cells broken bone cells by RT-PCR method; Western-blot method to detect the expression of RANK protein in the cell body of broken bone cells. Results: (1) RA group and healthy control group of osteoclasts morphologically similar, no 鏄庢樉鍖哄埆;RA緇勭牬楠ㄧ粏鑳?yōu)鏁伴嚕?

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