【摘要】： 目的核受體肝X受體(liver X receptor, LXR)α是在肝臟以及巨噬細(xì)胞中呈高表達(dá)的核受體,它作為諸多基因的轉(zhuǎn)錄因子在機(jī)體發(fā)揮著重要作用。許多實(shí)驗(yàn)表明LXRα作為一個(gè)轉(zhuǎn)錄開關(guān),除了誘導(dǎo)許多參與膽固醇逆轉(zhuǎn)動(dòng)、肝糖原代謝以及脂肪酸合成的基因外,另外LXRα還抑制了一系列由細(xì)菌感染、LPS、TNF-α或IL-1β等因子誘導(dǎo)的炎癥基因。但LXRα負(fù)性調(diào)控炎性基因的表達(dá)的具體機(jī)制尚不明確。本實(shí)驗(yàn)旨在通過觀察LXRα激動(dòng)劑對LPS刺激后Kupffer細(xì)胞IRF3、GRIP1、LXRα及炎癥因子IFNβ表達(dá)的影響,探討LXRα抑制LPS誘導(dǎo)的Kupffer細(xì)胞(KCs)激活效應(yīng)的可能機(jī)制。 方法采用密度梯度離心法結(jié)合選擇性貼壁法分離雄性KM小鼠肝臟中的Kupffer細(xì)胞,分離出的Kupffer細(xì)胞用含20%FCS的RPMI1640培養(yǎng)液24h后隨機(jī)分為四組:(1)正常對照組:以RPMI1640完全培養(yǎng)液置孵箱培養(yǎng)30h;(2)LPS處理組:倒掉原培養(yǎng)液,以RPMI1640完全培養(yǎng)液培養(yǎng)24h后,再用含1μg/ml LPS的RPMI1640完全培養(yǎng)液培養(yǎng)6h;(3)T0901317組:倒掉原培養(yǎng)液,用含5μg/ml T0901317的RPMI1640完全培養(yǎng)液培養(yǎng)24h,再以RPMI1640完全培養(yǎng)液培養(yǎng)6h;(4)LPS+T0901317組:倒掉原培養(yǎng)液,以含5μg/ml T0901317的RPMI1640完全培養(yǎng)液培養(yǎng)24h后,再改為含1μg/ml LPS的RPMI1640完全培養(yǎng)液,置孵箱培養(yǎng)6h。收集培養(yǎng)上清液及細(xì)胞,采用免疫細(xì)胞化學(xué)(SABC法)及Western blot法檢測Kupffer細(xì)胞的LXRα、GRIP1及IRF3蛋白表達(dá)水平;SYBR Green I嵌合熒光法Real-Time PCR測定LXRα、GRIP1及IRF3 mRNA表達(dá)水平;酶聯(lián)免疫吸附法(ELISA)檢測Kupffer細(xì)胞培養(yǎng)上清液中IFNβ含量。 結(jié)果(1)免疫細(xì)胞化學(xué)結(jié)果:LXRα、IRF3及GRIP1陽性表達(dá)均位于核內(nèi),陽性被染成棕色。IRF3蛋白在LPS處理組染色最深,聯(lián)合處理組次之,在正常對照組及LXR激動(dòng)劑幾乎未著色;GRIP1的染色情況與IRF3大致相同;LXRα陽性染色在LXRα激動(dòng)劑組表達(dá)最高,在聯(lián)合處理組明顯降低,在LPS處理組最低。(2)以各指標(biāo)的log cDNA/logβ-actin比值的x±s表示各指標(biāo)mRNA平均表達(dá)水平。在LPS組中IRF3及GRIP1 mRNA表達(dá)量最高(分別為1.089±0.0074、0.8922±0.0095),聯(lián)合處理組中其表達(dá)明顯降低(分別為0.7234±0.0072、0.6905±0.0042),兩組間均數(shù)差異均有顯著意義(P0.05);在LPS組及聯(lián)合處理組中,IRF3及GRIP1 mRNA水平均高于對照組(分別為0.3558±0.0051、0.3842±0.0083 )及LXRα激動(dòng)劑組(分別為0.333±0.0054、0.2778±0.0091),差異具有統(tǒng)計(jì)學(xué)意義(P0.05);LXRα在LPS組中(0.2722±0.0038)的表達(dá)明顯低于其它三組(對照組:0.3953±0.0051,聯(lián)合處理組:0.7963±0.0075,LXRα激動(dòng)劑組:0.9912±0.0098),差異具有統(tǒng)計(jì)學(xué)意義(P0.05);LXRα在LXRα激動(dòng)劑組的表達(dá)最高,與其它三組相比具有統(tǒng)計(jì)學(xué)意義(P0.05);聯(lián)合處理組中,LXRα表達(dá)明顯低于LXRα激動(dòng)劑組(P0.05),但又顯著高于其它兩組(P0.05)。(3)Western blot結(jié)果:用內(nèi)參照對待測物灰度值進(jìn)行標(biāo)準(zhǔn)校正。IRF3及GRIP1蛋白表達(dá)量在LPS組最高(分別為0.388±0.018, 0.276±0.015),聯(lián)合處理組表達(dá)明顯降低(分別為0.318±0.014, 0.224±0.017),兩組間均數(shù)差異均有顯著意義(P0.05);在LPS組及聯(lián)合處理組中,IRF3及GRIP1蛋白表達(dá)量均高于對照組(分別為0.268±0.025、0.162±0.013)及LXRα激動(dòng)劑組(分別為0.213±0.017、0.133±0.013),差別具有統(tǒng)計(jì)學(xué)意義(P0.05); LPS組LXRα表達(dá)最低(其值為0.534±0.014),明顯低于其它三組(對照組:1.03±0.024,聯(lián)合處理組:1.224±0.027,LXRα激動(dòng)劑組:1.74±0.034) ,差別具有統(tǒng)計(jì)學(xué)意義(P0.05);LXRα在LXRα激動(dòng)劑組的表達(dá)最高,與其它三組相比具有統(tǒng)計(jì)學(xué)意義(P0.05);聯(lián)合處理組中,LXRα表達(dá)明顯低于LXRα激動(dòng)劑組(P0.05),但又顯著高于其它兩組(P0.05)。(4)ELISA結(jié)果表明:IFNβ在LPS處理組的含量(329.5±35)顯著高于對照組(129.6±17)及LXRα激動(dòng)劑組(112.8±24),差異具有統(tǒng)計(jì)學(xué)意義(P0.05);聯(lián)合處理組IFNβ含量(224.4±33)比LPS處理組明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05);IFNβ表達(dá)LXRα激動(dòng)劑組表達(dá)最低。 結(jié)論本實(shí)驗(yàn)結(jié)果表明,在應(yīng)用LPS處理之前預(yù)防性應(yīng)用LXRα激動(dòng)劑,能明顯抑制Kupffer細(xì)胞的IRF3及GRIP1表達(dá),且在應(yīng)用LXRα激動(dòng)劑后LPS所誘生的炎癥因子IFNβ大大降低,進(jìn)一步說明了LXRα對IRF3通路的抑制。此外,相對于對照組及LXRα激動(dòng)劑組,在經(jīng)過LPS處理后,LXRα的表達(dá)明顯降低,IFNβ表達(dá)明顯上升。因些,LXRα與IRF3存在相互抑制作用,LXRα能夠通過抑制IRF3、GRIP1的表達(dá)而發(fā)揮抗炎效應(yīng),從而抑制LPS所誘導(dǎo)的Kupffer細(xì)胞活化。
[Abstract]:Objective Liver X receptor (LXR) alpha is a highly expressed nuclear receptor in the liver and macrophages. It plays an important role as a transcription factor for many genes in the body. In addition, LXRalpha inhibited a series of inflammatory genes induced by bacterial infection, LPS, TNF-alpha or IL-1bet. However, the mechanism of LXRalpha negatively regulating the expression of inflammatory genes remains unclear. To explore the possible mechanism of LXR alpha inhibiting LPS induced Kupffer cell (KCs) activation.
Methods Kupffer cells were isolated from the liver of male KM mice by density gradient centrifugation combined with selective adherence method. The isolated Kupffer cells were randomly divided into four groups after 24 hours in RPMI 1640 medium containing 20% FCS: (1) normal control group: incubated in RPMI 1640 incubator for 30 hours; (2) LPS treatment group: the original culture medium was poured out and RPMI 1640 was used to complete the incubation. T0901317 group: the original medium was removed, the RPMI1640 complete medium containing 5 ug/ml T0901317 was cultured for 24 hours, and then the RPMI1640 complete medium containing 5 ug/ml T0901317 was cultured for 6 hours; (4) LPS+T0901317 group: the original medium was poured out and the RPMI1640 complete medium containing 5 ug/ml T0901317 was cultured for 6 hours; (4) LPS+T0901317 group: the original medium was poured out and the RPMI1640 complete medium containing 5 ug/ml T0901317 was cultured. The culture supernatant and cells were collected and the expression levels of LXR alpha, GRIP1 and IRF3 were detected by immunocytochemistry (SABC) and Western blot. The expression levels of LXR alpha, GRIP1 and IRF3 mRNA in Kupffer cells were detected by SYBR Green I chimeric fluorescence Real-Time PCR. Enzyme linked immunosorbent assay (ELISA) was used to detect the content of IFN beta in Kupffer cell supernatant.
Results (1) Immunocytochemical staining showed that LXRalpha, IRF3 and GRIP1 were positively expressed in the nucleus, and the positive staining was brown. IRF3 protein stained the deepest in the LPS treatment group, followed by the combined treatment group, in the normal control group and LXR agonist almost no staining; GRIP1 staining was approximately the same as IRF3; LXRalpha positive staining was the most expressed in the LXRalpha agonist group. The expression of IRF3 and GRIP1 mRNA was the highest in LPS group (1.089.0074, 0.8922.0095, respectively). The expression of IRF3 and GRIP1 mRNA was significantly decreased in LPS group (0.7234.0072, 0.690, respectively). The levels of IRF3 and GRIP1 mRNA in LPS group and combined treatment group were higher than those in control group (0.3558.0051, 0.3842.0083) and LXR alpha agonist group (0.333.0054, 0.2778.0091, respectively), the difference was statistically significant (P 0.05). The expression of LXRalpha was significantly lower than that of the other three groups (control group: 0.3953 + 0.0051, combined treatment group: 0.7963 + 0.0075, LXRalpha agonist group: 0.9912 + 0.0098), the difference was statistically significant (P 0.05); the expression of LXRalpha in LXRalpha agonist group was the highest, compared with the other three groups was statistically significant (P 0.05); the expression of LXRalpha in combined treatment group was significantly lower than that in LXRalpha agonist group (P 0.05). The expression of IRF3 and GRIP1 protein was the highest in LPS group (0.388 [0.018] and 0.276 [0.015] respectively. The expression of IRF3 and GRIP1 protein was significantly lower in combined treatment group (0.318 [0.014] and 0.224 [0.017] respectively). The expression of IRF3 and GRIP1 protein in LPS group and combined treatment group were higher than those in control group (0.268.025, 0.162.013) and LXR alpha agonist group (0.213.017, 0.133.013, respectively), the difference was statistically significant (P 0.05); the expression of LXR alpha in LPS group was the lowest (0.534.014), significantly lower than that in control group (0.014). The expression of LXRalpha in the three groups (control group: 1.03 + 0.024, combined treatment group: 1.224 + 0.027, LXRalpha agonist group: 1.74 + 0.034) was statistically significant (P 0.05); the expression of LXRalpha in the LXRalpha agonist group was the highest, compared with the other three groups was statistically significant (P 0.05); the expression of LXRalpha in the combined treatment group was significantly lower than that in the LXRalpha agonist group (P 0.05). The results of ELISA showed that the content of IFN-beta in LPS group (329.5+35) was significantly higher than that in control group (129.6+17) and LXR-alpha agonist group (112.8+24), the difference was statistically significant (P 0.05); the content of IFN-beta in combined treatment group (224.4+33) was significantly lower than that in LPS group (P 0.05); The agonist group showed the lowest expression.
Conclusion LXR-alpha agonist can significantly inhibit the expression of IRF3 and GRIP1 in Kupffer cells before LPS treatment, and the expression of IFN-beta induced by LPS is significantly decreased after LXR-alpha agonist treatment, which further illustrates the inhibition of LXR-alpha on IRF3 pathway. After LPS treatment, the expression of LXRalpha was significantly decreased and the expression of IFNbeta was significantly increased. Therefore, LXRalpha and IRF3 were mutually inhibited. LXRalpha could exert anti-inflammatory effects by inhibiting the expression of IRF3 and GRIP1, thereby inhibiting the activation of Kupffer cells induced by LPS.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R363

【共引文獻(xiàn)】

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