【摘要】：目的:白細(xì)胞介素18(IL-18)和白細(xì)胞介素12(IL-12)是具有多種生物學(xué)功能的炎癥因子,近來(lái)發(fā)現(xiàn)兩者在動(dòng)脈粥樣硬化(As)斑塊中過(guò)量表達(dá)。本文觀察IL-18和IL-12對(duì)THP-1巨噬細(xì)胞源性泡沫細(xì)胞ATP結(jié)合盒轉(zhuǎn)運(yùn)體A1(ABCA1)表達(dá)和膽固醇流出的影響,并探討兩者的協(xié)同效應(yīng)及其調(diào)節(jié)ABCA1介導(dǎo)的膽固醇流出的相關(guān)機(jī)制。 方法:THP-1巨噬細(xì)胞源性泡沫細(xì)胞經(jīng)不同濃度IL-18(0,2,20,200 ng/ml)或IL-12(0,0.2,2,20 ng/ml)處理或者用2ng/ml IL-12和20 ng/ml IL-18共處理不同時(shí)間(0,6,12,24h);運(yùn)用液體閃爍計(jì)數(shù)器檢測(cè)細(xì)胞內(nèi)膽固醇流出,高效液相色譜分析細(xì)胞內(nèi)總膽固醇、游離膽固醇和膽固醇酯含量,實(shí)時(shí)熒光定量PCR檢測(cè)IL-18R、IL-12R、ABCA1和LXRαmRNA表達(dá)情況,Western印跡法檢測(cè)IL-18R、ABCA1、LXRα及核內(nèi)NF-κB p65蛋白質(zhì)的表達(dá)水平。以IL-18R抑制劑IL-18結(jié)合蛋白(IL-18BP)和NF-κB抑制劑對(duì)甲苯磺酰-L-苯丙氨酸氯甲基甲酮(N-p-Tosyl-L-phenylalanine chloro- methyl ketone, TPCK)預(yù)處理細(xì)胞3h再加入IL-18/IL-12共處理細(xì)胞24小時(shí),觀察對(duì)ABCA1及其介導(dǎo)的膽固醇流出的影響。 結(jié)果:IL-18和IL-12單獨(dú)處理THP-1巨噬細(xì)胞源性泡沫細(xì)胞并不明顯影響細(xì)胞總膽固醇、游離膽固醇、膽固醇酯含量及細(xì)胞內(nèi)膽固醇流出,但I(xiàn)L-12預(yù)處理或IL-18/IL-12共處理后呈濃度和時(shí)間依賴性抑制ABCA1 mRNA及蛋白質(zhì)的表達(dá),核內(nèi)NF-κB p65蛋白質(zhì)的表達(dá)水平增加,用IL-18BP和TPCK預(yù)處理細(xì)胞后,IL-18/IL-12的這種作用被抑制。IL-12促進(jìn)IL-18R表達(dá),但LXRαmRNA和蛋白質(zhì)的表達(dá)均不受IL-18/IL-12的影響。 結(jié)論:IL-18/IL-12共刺激下調(diào)THP-1巨噬細(xì)胞源性泡沫細(xì)胞ABCA1的表達(dá)和細(xì)胞內(nèi)膽固醇流出,促進(jìn)細(xì)胞內(nèi)脂質(zhì)蓄積,IL-18R和NF-κB信號(hào)途徑介導(dǎo)了IL-18對(duì)ABCA1的抑制作用,而IL-12通過(guò)促進(jìn)泡沫細(xì)胞IL-18R表達(dá)參與了IL-18對(duì)ABCA1的抑制作用。
[Abstract]:Aim: interleukin-18 (IL-18) and interleukin-12 (IL-12) are inflammatory factors with many biological functions. Recently, it has been found that both of them are overexpressed in atherosclerotic (As) plaques. The effects of IL-18 and IL-12 on the expression of ATP binding cassette transporter A1 (ABCA1) and cholesterol efflux in THP-1 macrophage derived foam cells were studied. Methods: THP-1 macrophage derived foam cells were treated with different concentrations of IL-18 (0 ~ 220200 ng/ml) or IL-12 (0 ~ 0.2o ~ (22) ~ 20 ng/ml) or co-treated with 2ng/ml IL-12 and 20 ng/ml IL-18 for different time (0 ~ 61212 ~ 24 h), and cholesterol efflux was detected by liquid scintillation counter. The contents of total cholesterol, free cholesterol and cholesterol ester were analyzed by HPLC, the expression of IL-18R,IL-12R,ABCA1 and LXR 偽 mRNA was detected by real-time fluorescence quantitative PCR, and the expression of IL-18R,ABCA1,LXR 偽 and NF- 魏 B p65 protein was detected by Western blot. IL-18R inhibitor IL-18 binding protein (IL-18BP) and NF- 魏 B inhibitor p-toluenesulfonyl-L-phenylalanine ketone (N-p-Tosyl-L-phenylalanine chloro- methyl ketone, TPCK) were pretreated with N-p-Tosyl-L-phenylalanine chloro- methyl ketone, TPCK) for 3 h and then co-treated with IL-18/IL-12 for 24 hours. The effects of N-p-Tosyl-L-phenylalanine chloro- methyl ketone, TPCK) on ABCA1 and cholesterol efflux were observed. Results: IL-18 and IL-12 alone did not affect the total cholesterol, free cholesterol, cholesterol ester content and intracellular cholesterol efflux of THP-1 macrophage derived foam cells. However, IL-12 pretreatment or co-treatment with IL-18/IL-12 inhibited the expression of ABCA1 mRNA and protein in a concentration-and time-dependent manner, and the expression of NF- 魏 B p65 increased in nucleus. After pretreatment with IL-18BP and TPCK, this effect of IL-18/IL-12 was inhibited. IL-12 promoted IL-18R expression. However, the expression of LXR 偽 mRNA and protein was not affected by IL-18/IL-12. Conclusion: IL-18/IL-12 costimulation down-regulates the expression of ABCA1 and intracellular cholesterol efflux in THP-1 macrophage derived foam cells, and promotes intracellular lipid accumulation. IL-18R and NF- 魏 B signaling pathway mediate the inhibitory effect of IL-18 on ABCA1. IL-12 plays an important role in the inhibition of ABCA1 by promoting the expression of IL-18R in foam cells.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R363

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