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糖皮质激素诱导的肿瘤坏死因子配体调控炎症反应的机制

Mechanisms of glucocorticoid-induced tumor necrosis factor ligand in regulating the inflammatory reaction

摘要:

目的 探讨库普弗细胞中糖皮质激素诱导的肿瘤坏死因子配体(GITRL)调控炎症反应的机制.方法 分离小鼠库普弗细胞和T细胞,转染库普弗细胞并与T细胞共培养,将共培养细胞分为6组:空白对照组,共培养细胞只加DMEM培养基;大肠埃希杆菌脂多糖(LPS)组,共培养细胞培养基中加入1 mg/L的LPS;沉默组、沉默对照组、质粒转染组、空载体质粒组,各组的库普弗细胞分别转染GITRLsiRNA、control siRNA、pEGFP-N1 GITRL、pEGFP-N1 control后与T细胞共培养后再加入LPS(1 mg/L),置孵箱培养24 h后处理.细胞免疫荧光法和Western blot法分别检测库普弗细胞的GITRL和程序性死亡配体(PDL1)的表达,MTT法和Annexin V/FITC染色流式分析仪分别检测T细胞增殖和凋亡,ELISA法检测上清液TNFα的含量.数据分析采用独立样本t检验和单因素方差分析(N-K检验).结果 转染24 h后荧光显微镜观察转染细胞荧光强度初步判断转染GITRL siRNA和转染pEGFP-N1 GITRL的库普弗细胞转入效率分别为90%和85%.转染GITRL siRNA的库普弗细胞GITRL蛋白表达较正常库普弗细胞显著下降,而转染pEGFP-N1 GITRL的库普弗细胞GITRL蛋白表达较正常库普弗细胞显著升高(=41.72,13.10,P<0.05);各转染对照库普弗细胞的GITRL蛋白表达与正常库普弗细胞比较,差异无统计学意义(F=2.27,P>0.05).LPS组GITRL荧光强度明显高于空白对照组(t=49.29,P<0.05);与LPS组比较,沉默组GITRL的表达明显下降(t =9.84,P<0.05);质粒转染组中GITRL的表达明显增加(=5.78,P<0.05);各转染对照组(沉默对照组、空载体质粒组)中GITRL的表达与LPS组比较,差异无统计学意义(F=0.86,P>0.05).LPS组PDL1蛋白的表达与空白对照组比较明显下降(t=18.83,P<0.05);与LPS组比较,质粒转染组中PDL1蛋白的表达下降更明显(t=11.79,P<0.05);沉默组PDL1蛋白的表达则介于LPS组和质粒转染组之间(t=19.08,P<0.05);各转染对照组(沉默对照组、空载体质粒组)与LPS组比较,差异无统计学意义(F=2.22,P>0.05).LPS组与空白对照组比较,T细胞增殖明显增加,凋亡显著减少(t=49.43,40.11,P<0.05);与LPS组比较,质粒转染组T细胞增殖和凋亡表现为更明显的相同趋势(t=5.77,12.64,P<0.05);而沉默组T细胞的增殖减少而凋亡明显增加(t=17.00,49.90,P<0.05);各转染对照组(沉默对照组、空载体质粒组)与LPS组比较,差异无统计学意义(F=1.87,1.35,P>0.05).LPS组与空白对照组比较,TNF-α的表达明显增加(t=125.68,P<0.05);与LPS组比较,沉默组TNF-α显著下降(t=119.65,P<0.05);质粒转染组TNF-α则显著增加(t=147.70,P<0.05);各转染对照组(沉默对照组和空载体质粒组)与LPS组比较,差异无统计学意义(F=0.14,P>0.05).结论 库普弗细胞通过上调GITRL的表达抑制PDL1,激活T细胞增殖,促进炎症反应;干扰GITRL的表达可恢复免疫平衡,抑制炎症反应.

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abstracts:

Objective To investigate the role of glucocorticoid-induced tumor necrosis factor ligand (GITRL) in regulating the inflammatory reaction of kupffer cells.Methods The kupffer cells and T cells of mice were isolated and divided into 6 groups after being co-cultured:control group,kupffer cells and T cells were cultured in DMEM only; lipopolysaccharide (LPS) group,kupffer cells and T cells were cultured in media with LPS (1 mg/L) ; LPS + GITRL siRNA group,kupffer cells and T cells were cultured in media as the LPS group after transfected with GITRL siRNA ; LPS + control siRNA group,kupffer cells and T cells were cultured in media as the LPS group after transfected with control siRNA; LPS + pEGFP-N1 GITRL group,kupffer cells and T cells were cultured in media as the LPS group after transfected with pEGFP-N1 GITRL plasmid; LPS + pEGFP-N1 control group,kupffer cells and T cells were cultured in media as the LPS group after transfected with control plasmid.After 24 hours of treatment,the expressions of GITRL and PDL1 of kupffer cells were detected by immunofluorescence and western blot,respectively.The proliferation and apoptosis of T cells were measured by MTF assay and Annexin V/PI flow cytometry,respectively.The expression of tumor necrosis factor (TNF-α) in the supernatant fluid was measured by ELISA.All data were analyzed using the independent t test and one-way analysis of variance.Results The transfection efficiencies of GITRL siRNA and pEGFP-N1 GITRL were 90% and 85%,respectively.Compared with normal kupffer cells,the protein expression of GITRL of kupffer cells transfected with GITRL siRNA was significantly decreased,while the protein expression of GITRL of kupffer cells transfected with pEGFP-N1 GITRL was significantly increased (t =41.72,13.10,P < 0.05).There was no significant difference in the protein expressions of GITRL between normal kupffer cells and those in the control groups (F =2.27,P > 0.05).The fluorescence intensity of GITRL in the LPS group was significantly higher than that in the control group (t =49.29,P < 0.05).Compared with LPS group,the activation of GITRL expression by the LPS was significantly suppressed in the LPS + GITRL siRNA group (t =9.84,P < 0.05),while the expression of GITRL in the LPS + pEGFP-N1 GITRL group was significantly increased (t =5.78,P < 0.05).There was no significant difference in the GITRL expression among the LPS + control siRNA group,LPS + pEGFP-N1 control group and LPS group (F =0.86,P > 0.05).The expression of PDL1 in the LPS group was significantly lower than that in the control group (t =18.83,P <0.05).Compared with LPS group,the expression of PDL1 in the LPS + pEGFP-N1 GITRL group was significantly suppressed (t =11.79,P < 0.05),while the expression of PDL1 in the LPS + GITRL siRNA group was significantly stronger (t =19.08,P < 0.05).There was no significantly difference in the expression of PDL1 in the LPS + control siRNA group,LPS + pEGFP-N1 control group and LPS group (F =2.22,P > 0.05).The proliferation of T cells was increased and the number of apoptotic T cell was decreased in the LPS group when compared with control group (t =49.43,40.11,P < 0.05).Compared with LPS group,the proliferation and apoptosis of T cells in the LPS + pEGFP-N1 GITRL group had the same trend (t =5.77,12.64,P <0.05); while the proliferation of T cells was decreased and the apoptosis of T cells was increased in the LPS + GITRL siRNA group (t =17.00,49.90,P < 0.05).There was no significant difference in the proliferation and apoptosis of T cells among the LPS + control siRNA group,LPS + pEGFP-N1 control group and LPS group (F =1.87,1.35,P > 0.05).The expression of TNF-α was significantly higher in the LPS group than that in the control group (t =125.68,P < 0.05).Compared with the LPS group,the expression of TNF-α was significantly decreased in the LPS + GITRL siRNA group (t =119.65,P < 0.05),while the expression of TNF-α in the LPS + pEGFP-N1 GITRL group was significantly increased (t =147.70,P < 0.05).There was no significant difference in the TNF-α expression among the LPS + control siRNA group,LPS + pEGFP-N1 control group and LPS group (F =0.14,P > 0.05).Conclusion Kupffer cells suppress the expression of PDL1 by upregulating GITRL,and thus activate the proliferation of T cells and promote the inflammatory reaction.The immunologic balance may be recovered after the interference of GITRL to restrain the inflammatory reaction.

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