【摘要】： 目的:應(yīng)用TLR4特異性激動劑脂多糖(LPS)刺激體外培養(yǎng)的人單核細(xì)胞,激活TLR4/NF-κB信號途徑,將含有A20基因的質(zhì)粒轉(zhuǎn)染入單核細(xì)胞進(jìn)行干預(yù),觀察A20過表達(dá)后單核細(xì)胞膜受體TLR4表達(dá)的變化,以及對下游促炎因子TNF-α、IL-12及抗炎因子IL-10表達(dá)的調(diào)節(jié),并進(jìn)一步研究A20過表達(dá)對促炎因子/抗炎因子即TNF-α/ IL-10和IL-12/ IL-10比率的影響,以探討鋅脂蛋白A20對單核細(xì)胞炎癥反應(yīng)的保護(hù)作用及可能的調(diào)節(jié)機(jī)制。 方法:外周血經(jīng)EDTA抗凝,Ficoll細(xì)胞分離液分離外周血單個核細(xì)胞,將單核細(xì)胞貼壁培養(yǎng)于不含血清的RPMI-1640培養(yǎng)液中,調(diào)整單核細(xì)胞濃度為1×105個/ml,實(shí)驗(yàn)設(shè)A組(為空白對照組,培養(yǎng)液中不加任何干預(yù)因素);B組(為LPS組,無血清培養(yǎng)48小時,在培養(yǎng)基中加入1mg/L的LPS作用24小時); C組(為A20轉(zhuǎn)染組,無血清培養(yǎng)24小時,轉(zhuǎn)染pCAGGS-GFP/A20質(zhì)粒陽離子脂質(zhì)體復(fù)合物培養(yǎng)48小時); D組(為LPS+A20轉(zhuǎn)染組,無血清培養(yǎng)24小時,轉(zhuǎn)染pCAGGS-GFP/A20質(zhì)粒陽離子脂質(zhì)體復(fù)合物培養(yǎng)48小時,于轉(zhuǎn)染過程中加入1mg/L的LPS作用24小時)。胰酶消化法收集以上各組單核細(xì)胞;采用免疫熒光方法檢測GFP報告基因,免疫組化檢測A20蛋白的表達(dá);提取總mRNA用RT-PCR檢測內(nèi)源性A20、外源性A20及TLR4的mRNA表達(dá);應(yīng)用流式細(xì)胞檢測技術(shù)檢測TLR4的表達(dá),以CD14+為單核細(xì)胞標(biāo)記,采用未轉(zhuǎn)染的細(xì)胞進(jìn)行調(diào)零,記錄熒光陽性細(xì)胞的百分率;采用雙抗體夾心ELISA方法檢測上清液TNF-α、IL-12及IL-10表達(dá)水平。 結(jié)果: 1.單核細(xì)胞受到LPS(1mg/L)刺激后,其自身受體TLR4和內(nèi)源性A20的mRNA和蛋白表達(dá)以及促炎因子TNF-α、IL-12和抗炎因子IL-10表達(dá)較對照組均明顯升高;TNF-α/IL-10和IL-12/IL-10的比率均明顯高 于對照組。2.轉(zhuǎn)染A20基因的單核細(xì)胞,在無LPS刺激的條件下,其自身受體TLR4和內(nèi)源性A20的mRNA和蛋白表達(dá)以及促炎因子TNF-α、IL-12和抗炎因子IL-10的表達(dá)與對照組相比均無明顯變化;TNF-α/IL-10和IL-12/IL-10的比率與對照組相比也無明顯變化。 3.轉(zhuǎn)染A20基因的單核細(xì)胞在受到LPS(1mg/L)刺激后,其自身受體TLR4mRNA和蛋白表達(dá)以及促炎因子TNF-α、IL-12的表達(dá)均顯著低于LPS組,而抗炎因子IL-10的表達(dá)明顯上調(diào),高于對照組和LPS組;而TNF-α/IL-10和IL-12/IL-10的比率明顯下降,低于LPS組。 結(jié)論: 1.TLR4激活介導(dǎo)單核細(xì)胞的炎癥反應(yīng),且正反饋調(diào)節(jié)其自身受體TLR4和內(nèi)源性A20的表達(dá)上調(diào);A20參與單核細(xì)胞TLR4激活所介導(dǎo)的炎癥反應(yīng),其表達(dá)增加與TLR4表達(dá)的增加有關(guān)。 2.單純提高A20表達(dá)對未被激活的單核細(xì)胞TLR4及其信號通路影響不大,提示A20不直接引起炎癥反應(yīng),其作用具有炎癥或TLR4活化依賴性。 3.A20過表達(dá)可抑制TLR4激活所介導(dǎo)的單核細(xì)胞的炎癥反應(yīng),其機(jī)制是負(fù)反饋抑制TLR4的表達(dá),進(jìn)而抑制促炎因子的表達(dá),增加抗炎因子的表達(dá),改善促炎因子/抗炎因子的平衡關(guān)系,從而達(dá)到抑制炎癥反應(yīng)的作用。 4.本研究提示:通過基因轉(zhuǎn)染增加A20的表達(dá),可抑制炎癥過程中單核細(xì)胞參與的炎癥反應(yīng),該研究為基因治療動脈粥樣硬化等炎癥性疾病提供了重要的理論和實(shí)踐依據(jù)。
[Abstract]:Objective: To stimulate the human monocytes cultured in vitro by TLR4 specific agonist lipopolysaccharide (LPS), activate the signaling pathway of TLR4/ NF-B, and transfect the plasmid containing the A20 gene into the monocytes for intervention, and observe the changes of the expression of the receptor TLR4 in the mononuclear cell membrane after the overexpression of A20. as well as the regulation of the expression of the downstream proinflammatory factor TNF-1, IL-12 and the anti-inflammatory factor IL-10, and further study the effect of the overexpression of A20 on the pro-inflammatory factor/ anti-inflammatory factor, that is, the TNF-1/ IL-10 and the IL-12/ IL-10 ratio, In order to study the protective effect of the zinc-lipoprotein A20 on the inflammatory reaction of monocytes and the possible regulatory mechanism. Methods: The peripheral blood mononuclear cells were isolated from peripheral blood by EDTA and Ficoll cell. The mononuclear cells were cultured in RPMI-1640 medium without serum. The concentration of mononuclear cells was 1-105/ ml. Group B (no intervention factor in the culture solution); Group B (for LPS group, no serum culture for 48 hours, add 1 mg/ L of LPS for 24 hours in culture medium); Group C (for A20 transfection group, no serum culture for 24 hours, transfection of pCAGGS-GFP/ A20 plasmid cationic liposome complex culture 48 small 1 mg/ L of LPS was added to the transfected pCAGGS-GFP/ A20 plasmid pCAGGS-GFP/ A20 plasmid for 48 hours. The expression of endogenous A20, exogenous A20 and TLR4 was detected by RT-PCR and the expression of endogenous A20, exogenous A20 and TLR4 was detected by RT-PCR. Expression, labeling with CD14 + as a monocyte, zeroing with untransfected cells, recording the percentage of fluorescent positive cells, and detecting the expression of TNF-1, IL-12 and IL-10 in the supernatant using a double-antibody sandwich ELISA method. Level. Results:1. After stimulation by LPS (1 mg/ L), the expression of the mRNA and protein of the self-receptor TLR4 and endogenous A20 and the expression of the pro-inflammatory factor, TNF-1, IL-12 and IL-10 in the control group, were significantly higher than that in the control group, and TNF-1/ IL-10 and IL-12/ IL-10. The ratio of 10 is clear 2. The expression of the mRNA and protein of the autoreceptor TLR4 and the endogenous A20 and the expression of the pro-inflammatory factor TNF-1, IL-12 and the anti-inflammatory factor IL-10 in the absence of LPS-stimulated monocytes in the control group. There was no significant change in the control group; the ratio of TNF-1/ IL-10 and IL-12/ IL-10 to 3. The expression of anti-inflammatory factor IL-10 was significantly lower than that of LPS group, and the expression of anti-inflammatory factor IL-10 was significantly lower than that of LPS group after LPS (1 mg/ L) was stimulated by LPS (1 mg/ L). Up-regulated, higher than control and LPS groups; TNF-1/ IL-10 and IL-12/ IL-10 ratio Conclusion:1. TLR4 activates the inflammatory response of monocytes, and positive feedback regulates the expression of its autoreceptor TLR4 and endogenous A20, and A20 is involved in the inflammation mediated by the activation of TLR4. 2. The expression of A20 is related to the increase of TLR4 expression. 3. The overexpression of A20 can inhibit the inflammatory reaction of the monocyte mediated by TLR4 activation, and its mechanism is negative feedback to inhibit the expression of TLR4, thereby inhibiting the expression of the pro-inflammatory factor. The expression of anti-inflammatory factor is increased, and the proinflammatory is improved. 4. The study suggests that the expression of A20 can be increased by gene transfection, and the inflammatory response of monocytes in the process of inflammation can be inhibited.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392.3

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 鄒瓊超;急性冠脈綜合征患者外周血單核細(xì)胞鋅指蛋白A20的表達(dá)及阿托伐他汀干預(yù)的研究[D];南華大學(xué);2012年

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本文編號：2499568