脂氧素A4在角质形成细胞及银屑病中的抗炎效应及分子机制研究

发布时间：2018-06-11 22:24

本文选题：角质形成细胞 + 炎症因子　；参考：《华中科技大学》2015年博士论文

【摘要】：第一部分：脂氧素A4通过调控SOCS2/TRAF6抑制脂多糖诱导的人角质形成细胞炎性因子的释放目的：研究脂氧素A4(LXA4)对脂多糖诱导的人角质形成细胞分泌细胞因子和趋化因子的作用效应及其可能上游机制。方法：分离培养正常人表皮角质形成细胞,首先逆转录PCR检测TLR4、脂氧素受体(ALXR)和芳香烃受体(AhR)在人角质形成细胞上的表达情况；实验分为三组：对照组.LPS(10ug/ml)组及LXA4(100nmol/L)+LPS组,分别作用相应的时间后,实时荧光定量PCR和ELISA分别从mRNA水平和蛋白水平检测各组角质形成细胞不同时间点TNF-a和IL-1β的表达量;实时荧光定量PCR和蛋白质印迹试验分别从mRNA水平和蛋白水平检测各组角质形成细胞SOCS2和TRAF6的表达；蛋白质印迹试验检测各组细胞细胞核中核转录因子NF.kB的表达量。结果：人角质形成细胞表达TLR4、脂氧素受体及芳香烃受体；从mRNA水平和蛋白水平,LXA4明显抑制LPS刺激诱导的TNF-a、IL-1β及TRAF6在角质形成细胞中的高表达,上调LPS干预下SOCS2的低表达；LXA4抑制LPS诱导的角质形成细胞中NF-κB的核转位。结论：LXA4可能通过上调SOCS2降解TRAF6抑制脂多糖刺激诱导的角质形成细胞TNF-a和IL-1p的表达。第二部分：脂氧素A4通过ERK/NF-kB信号通路抑制人角质形成细胞增殖及炎性因子/趋化因子的释放目的：研究在未激活及脂多糖刺激活化下的人角质形成细胞中,LXA4对细胞增殖和分泌炎性细胞因子及趋化因子的作用,探寻LXA4对角质形成细胞细胞周期和下游抗炎信号通路的机制研究。方法：实验分为四组,对照组、LXA4组(100noml/L)、LPS组(10ug/ml)和LXA4+LPS组。用WST-8和CFSE细胞示踪法检测细胞增殖及DNA染色流式细胞术检测细胞周期；实时荧光定量PCR和ELISA分别从mRNA水平和蛋白水平检测各组角质形成细胞细胞炎性因子和趋化因子的表达；实时荧光定量PCR和蛋白质印迹试验分别从mRNA水平和蛋白水平检测cyclin D1和p16INK4A的表达；蛋白质印迹试验检测ERK1/2和核转录因子NF-kB的表达量。结果：对未激活及脂多糖刺激活化下的人角质形成细胞,LXA4抑制其细胞增殖及炎症细胞因子/趋化因子的释放；对角质形成细胞造成细胞周期G0/G1期阻滞；细胞周期蛋白cyclin D1的表达被LXA4抑制,同时促进p16INK4A分子的表达；ERK1/2的磷酸化和NF-kB-p65的核转位都被LXA4所抑制。结论：LXA4能抑制未激活及脂多糖刺激活化下人角质形成细胞增殖和细胞炎性因子的释放,这种效应可能是通过干预信号分子cyclin D1/P16INK4A. ERK1/2和NF-kB实现的。第三部分：脂氧素A4腹腔注射改善咪喹莫特诱导的小鼠银屑病样皮损目的：观察LXA4对咪喹莫特诱导的小鼠银屑病模型的干预作用。方法：30只雌性Balb/C小鼠,随机等分为正常对照组、模型组及LXA4组。采应用银屑病皮损面积和疾病严重程度评分标准(psoriasisarea and severity index, PASI)观测各组模型小鼠皮损变化；光镜下测量表皮层厚度；光镜下观察皮损组织形态学变化;采用流式细胞术检测各组小鼠脾脏细胞CD4+T细胞中Th1及Th17细胞的分化差异。结果：模型组小鼠背部皮肤出现红斑、鳞屑及皮损增厚;皮损组织病理表现为角化不全、中性粒细胞微脓肿及表皮棘层增厚；真皮大量炎症细胞浸润,脾脏Th17细胞比例上升。与模型组比较,LXA4组小鼠银屑病样皮损红斑、鳞屑及增厚症状缓解,PASI分数降低,表皮厚度显著降低,皮损中性粒细胞微脓疡基本消退,角质形成细胞增殖明显减轻,真皮浅层炎症细胞浸润减少,脾脏Th17细胞比例下调。结论：LXA4能通过促中性粒细胞消退、抑制表皮细胞过度增殖及调节T淋巴细胞分化改善咪喹莫特诱导的小鼠银屑病样皮损。
[Abstract]:Part I: lipoxygenin A4 inhibits lipopolysaccharide induced release of inflammatory cytokines in human keratinocytes by regulating SOCS2/TRAF6.
Objective: To study the effect of lipoxin A4 (LXA4) on lipopolysaccharide induced cytokine and chemokines in human keratinocytes and its possible upstream mechanism.
Methods: normal human epidermal keratinocytes were isolated and cultured. First, the expression of TLR4, lipoxygenin receptor (ALXR) and aromatic hydrocarbon receptor (AhR) in human keratinocytes were detected by reverse transcriptase PCR. The experiment was divided into three groups:
In the control group.LPS (10ug/ml) and the LXA4 (100nmol/L) +LPS group, the real-time fluorescence quantitative PCR and ELISA were used to detect the expression of TNF-a and IL-1 beta in different time points of keratinocytes from each group from mRNA level and protein level, respectively. Real time fluorescence quantitative PCR and protein blotting tests were examined from mRNA level and protein level respectively. The expression of SOCS2 and TRAF6 in keratinocytes was measured, and the expression of nuclear transcription factor NF.kB was detected by Western blot.
Results: human keratinocytes express TLR4, lipoxygenin receptor and aromatics receptor. From the level of mRNA and protein, LXA4 obviously inhibits the TNF-a induced by LPS, the high expression of IL-1 beta and TRAF6 in keratinocytes, up regulation of the low expression of SOCS2 under the intervention of LPS; LXA4 inhibits the nuclear transposition of NF- kappa of LPS induced keratinocytes.
Conclusion: LXA4 may inhibit the expression of TNF-a and IL-1p in keratinocytes stimulated by lipopolysaccharide by up regulating the degradation of TRAF6 by SOCS2.
The second part: lipoxygenin A4 inhibits keratinocyte proliferation and the release of inflammatory factors / chemokines through ERK/NF-kB signaling pathway.
Objective: To investigate the effect of LXA4 on the proliferation and secretion of inflammatory cytokines and chemokines in human keratinocytes under inactivation and lipopolysaccharide activation, and to explore the mechanism of LXA4 on the cell cycle of keratinocytes and the mechanism of the downstream anti-inflammatory signaling pathway.
Methods: the experiment was divided into four groups, the control group, the LXA4 group (100noml/L), the LPS group (10ug/ml) and the LXA4+LPS group. The cell proliferation was detected by WST-8 and CFSE cell tracer, and the cell cycle was detected by the flow cytometry with DNA staining. The real-time fluorescent quantitative PCR and ELISA were used to detect the inflammatory factors of the keratinocyte cells from the mRNA level and protein level, respectively. The expression of chemokine; real-time fluorescence quantitative PCR and protein blotting test were used to detect the expression of cyclin D1 and p16INK4A from mRNA level and protein level, and the expression of ERK1/2 and nuclear transcription factor NF-kB was detected by Western blot test.
Results: LXA4 inhibited the proliferation of cells and the release of inflammatory cytokines / chemokine in human keratinocytes which were unactivated and activated by lipopolysaccharide. The cell cycle blocked the cell cycle of keratinocytes; the expression of cyclin cyclin D1 was inhibited by LXA4, and the expression of p16INK4A molecules was promoted; ERK1/2 Phosphorylation and nuclear translocation of NF-kB-p65 were inhibited by LXA4.
Conclusion: LXA4 can inhibit the proliferation of human keratinocytes and the release of inflammatory cytokines from inactivation and lipopolysaccharide activation. This effect may be achieved by interfering with the signal molecule cyclin D1/P16INK4A. ERK1/2 and NF-kB.
The third part: lipoxygenin A4 intraperitoneal injection improves imiquimod induced psoriasis like lesions in mice.
Objective: To observe the effect of LXA4 on the murine model of psoriasis induced by imiquimod.
Methods: 30 female Balb/C mice were randomly divided into the normal control group, the model group and the LXA4 group. The skin lesions of psoriasis and the severity of disease severity score (psoriasisarea and severity index, PASI) were used to observe the changes of skin lesions in each model mice, the thickness of the cortex was measured under light microscope, and the morphological changes of skin lesions were observed under light microscope. The differentiation of Th1 and Th17 cells in the CD4+T cells of spleen cells of each group was detected by flow cytometry. Results: the skin of the model group had erythema, scaly and skin lesions, and the pathological features of the skin lesions were keratosis, neutrophils microabscess and the thickening of the epidermis, the infiltration of numerous inflammatory cells in the dermis, and the spleen Th The proportion of 17 cells increased. Compared with the model group, the erythema, scaly and thickening symptoms of psoriatic skin lesions in the LXA4 group were relieved, the PASI score was reduced, the thickness of the epidermis was significantly reduced, the neutrophils microabscess was basically subsided, the proliferation of keratinocytes was significantly reduced, the infiltration of the superficial dermal inflammatory cells decreased, and the proportion of Th17 cells in the spleen was down.
Conclusion: LXA4 can improve the psoriasis like skin lesions of mice induced by imiquimod by promoting neutrophil degeneration, inhibiting the overproliferation of epidermal cells and regulating the differentiation of T lymphocyte.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R758.63

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