本文選題：姜黃素 + 類風(fēng)濕關(guān)節(jié)炎�。� 參考：《南京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：目的:觀察姜黃素(Curcumin,Cur)對(duì)類風(fēng)濕關(guān)節(jié)炎(rheumatoid arthritis,RA)患者破骨細(xì)胞生成的影響,探索核因子-κB(nuclear factor kappa B,NF-κB)信號(hào)通路在此過(guò)程中的調(diào)節(jié)機(jī)制,為姜黃素應(yīng)用于RA骨破壞的防治提供理論依據(jù)。方法:(1)分離RA 患者外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cells,PBMCs)誘導(dǎo)培養(yǎng)為破骨細(xì)胞,在培養(yǎng)過(guò)程中給予不同濃度姜黃素進(jìn)行干預(yù);(2)分組:Control組、Cur 2.5 μM 組、Cur 5μ 組、Cur 10 μM 組,姜黃素的濃度分別為 0、2.5 μmol/L、5 μmol/L、10 μmol/L;(3)誘導(dǎo)培養(yǎng)14天后進(jìn)行抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色標(biāo)記成熟破骨細(xì)胞,并行細(xì)胞計(jì)數(shù);(4)Western Blot檢測(cè)各組細(xì)胞中IκBα、p-IκBα蛋白的表達(dá);(5)Western Blot檢測(cè)細(xì)胞漿和細(xì)胞核中NF-κB p65的表達(dá);(6)細(xì)胞免疫熒光染色法觀察NF-κB p65核易位。結(jié)果:(1)培養(yǎng)14天后,各組細(xì)胞均符合破骨細(xì)胞的形態(tài)學(xué)特征;(2)TRAP染色結(jié)果顯示,與Control組相比,Cur 2.5 μM組、Cur 5 μM組和Cur 10 μM組TRAP陽(yáng)性細(xì)胞數(shù)均明顯減少,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);(3)Western Blot結(jié)果顯示,與Control組相比,Cur 2.5 μM組、Cur 5 μM組和Cur 10 μ組IκBα的蛋白表達(dá)水平明顯升高,p-IκBα的表達(dá)水平明顯下降,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);(4)與Control組相比,Cur 2.5 μM組、Cur 5μ 組和Cur 10 μ組細(xì)胞漿中NF-κB p65的表達(dá)水平明顯升高,而細(xì)胞核中NF-κB p65的表達(dá)水平明顯下降,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);(5)細(xì)胞免疫熒光染色結(jié)果顯示,在未刺激狀態(tài)下NF-κB p65主要分布于細(xì)胞漿中;經(jīng)RANKL刺激后,NF-κB p65在細(xì)胞漿的表達(dá)減少,在細(xì)胞核的表達(dá)增加;經(jīng)5 μM姜黃素干預(yù)后,與RANKL刺激后相比,NF-κB p65在細(xì)胞漿的表達(dá)增加,在細(xì)胞核的表達(dá)減少。結(jié)論:姜黃素通過(guò)抑制IκBα的磷酸化和IκBα的降解,減少了 NF-κB p65的核易位,從而抑制NF-κB活化,進(jìn)而達(dá)到抑制RA患者破骨細(xì)胞生成的作用。
[Abstract]:Aim: to investigate the effect of curcuminia curcuminus on osteoclast formation in rheumatoid arthritis (RA) patients, and to explore the regulatory mechanism of NF- 魏 B(nuclear factor kappa BnNF- 魏 B signaling pathway in the process, and to provide theoretical basis for the application of curcumin in the prevention and treatment of RA bone failure. Methods the peripheral blood mononuclear cells (PBMCs) from patients with RA were isolated and induced to be osteoclasts. Different concentrations of curcumin were given in the process of culture. The concentration of curcumin was 2.5 渭 mol / L ~ 5 渭 mol / L ~ (5 渭 mol / L ~ (10) 渭 mol / L ~ (3). After 14 days of induction and culture, mature osteoclasts were stained with tartrate-resistant acid phosphatase TRAPs. The expression of I 魏 B 偽 -p-I 魏 B 偽 protein was detected by Western Blot. The expression of NF- 魏 B p65 in cytoplasm and nucleus was detected by Western Blot. The nuclear translocation of NF- 魏 B p65 was observed by cell immunofluorescence staining. Results after 14 days of culture, the cells in each group were in accordance with the morphological characteristics of osteoclasts. The results showed that compared with Control group, the number of TRAP positive cells in cur5 渭 M group and Cur 10 渭 M group decreased significantly compared with Control group. The difference was statistically significant (P 0.05). The results of Western Blot showed that compared with Control group, the protein expression level of cur5 渭 M group and Cur 10 渭 group significantly increased the expression level of p-I 魏 B 偽 in Cur 2.5 渭 M group and Cur 10 渭 group, and the expression level of p-I 魏 B 偽 decreased significantly. Compared with the Control group, the expression of NF- 魏 B p65 in the cytoplasm of Cur 2.5 渭 M group and Cur 10 渭 group was significantly increased, while the expression level of NF- 魏 B p65 in the nucleus decreased significantly. The results of immunofluorescence staining showed that NF- 魏 B p65 was mainly distributed in the cytoplasm under the condition of no stimulation, and the expression of NF- 魏 B p65 in the cytoplasm decreased and increased in the nucleus after RANKL stimulation. After treated with 5 渭 M curcumin, the expression of NF- 魏 B p65 in cytoplasm increased and decreased in nucleus after RANKL stimulation. Conclusion: curcumin can reduce the nuclear translocation of NF- 魏 B p65 by inhibiting the phosphorylation of I 魏 B 偽 and the degradation of I 魏 B 偽, thereby inhibiting the activation of NF- 魏 B and thus inhibiting osteoclast formation in RA patients.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R593.22

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