锌脂蛋白A20抑制TLR4介导的人单细胞炎症反应的实验研究
发布时间：2019-06-14 17:54
【摘要】： 目的:应用TLR4特异性激动剂脂多糖(LPS)刺激体外培养的人单核细胞,激活TLR4/NF-κB信号途径,将含有A20基因的质粒转染入单核细胞进行干预,观察A20过表达后单核细胞膜受体TLR4表达的变化,以及对下游促炎因子TNF-α、IL-12及抗炎因子IL-10表达的调节,并进一步研究A20过表达对促炎因子/抗炎因子即TNF-α/ IL-10和IL-12/ IL-10比率的影响,以探讨锌脂蛋白A20对单核细胞炎症反应的保护作用及可能的调节机制。 方法:外周血经EDTA抗凝,Ficoll细胞分离液分离外周血单个核细胞,将单核细胞贴壁培养于不含血清的RPMI-1640培养液中,调整单核细胞浓度为1×105个/ml,实验设A组(为空白对照组,培养液中不加任何干预因素);B组(为LPS组,无血清培养48小时,在培养基中加入1mg/L的LPS作用24小时); C组(为A20转染组,无血清培养24小时,转染pCAGGS-GFP/A20质粒阳离子脂质体复合物培养48小时); D组(为LPS+A20转染组,无血清培养24小时,转染pCAGGS-GFP/A20质粒阳离子脂质体复合物培养48小时,于转染过程中加入1mg/L的LPS作用24小时)。胰酶消化法收集以上各组单核细胞;采用免疫荧光方法检测GFP报告基因,免疫组化检测A20蛋白的表达;提取总mRNA用RT-PCR检测内源性A20、外源性A20及TLR4的mRNA表达;应用流式细胞检测技术检测TLR4的表达,以CD14+为单核细胞标记,采用未转染的细胞进行调零,记录荧光阳性细胞的百分率;采用双抗体夹心ELISA方法检测上清液TNF-α、IL-12及IL-10表达水平。 结果: 1.单核细胞受到LPS(1mg/L)刺激后,其自身受体TLR4和内源性A20的mRNA和蛋白表达以及促炎因子TNF-α、IL-12和抗炎因子IL-10表达较对照组均明显升高;TNF-α/IL-10和IL-12/IL-10的比率均明显高 于对照组。2.转染A20基因的单核细胞,在无LPS刺激的条件下,其自身受体TLR4和内源性A20的mRNA和蛋白表达以及促炎因子TNF-α、IL-12和抗炎因子IL-10的表达与对照组相比均无明显变化;TNF-α/IL-10和IL-12/IL-10的比率与对照组相比也无明显变化。 3.转染A20基因的单核细胞在受到LPS(1mg/L)刺激后,其自身受体TLR4mRNA和蛋白表达以及促炎因子TNF-α、IL-12的表达均显著低于LPS组,而抗炎因子IL-10的表达明显上调,高于对照组和LPS组;而TNF-α/IL-10和IL-12/IL-10的比率明显下降,低于LPS组。 结论: 1.TLR4激活介导单核细胞的炎症反应,且正反馈调节其自身受体TLR4和内源性A20的表达上调;A20参与单核细胞TLR4激活所介导的炎症反应,其表达增加与TLR4表达的增加有关。 2.单纯提高A20表达对未被激活的单核细胞TLR4及其信号通路影响不大,提示A20不直接引起炎症反应,其作用具有炎症或TLR4活化依赖性。 3.A20过表达可抑制TLR4激活所介导的单核细胞的炎症反应,其机制是负反馈抑制TLR4的表达,进而抑制促炎因子的表达,增加抗炎因子的表达,改善促炎因子/抗炎因子的平衡关系,从而达到抑制炎症反应的作用。 4.本研究提示:通过基因转染增加A20的表达,可抑制炎症过程中单核细胞参与的炎症反应,该研究为基因治疗动脉粥样硬化等炎症性疾病提供了重要的理论和实践依据。
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.3
本文编号：2499568
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.3
【引证文献】
相关硕士学位论文 前1条
1 邹琼超;急性冠脉综合征患者外周血单核细胞锌指蛋白A20的表达及阿托伐他汀干预的研究[D];南华大学;2012年
，本文编号：2499568
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