C3aR激活在尿酸或脂多糖介导的肾小管上皮细胞CC类趋化因子表达中的作用
发布时间：2018-09-18 07:19
【摘要】：目的：探讨人肾皮质近曲小管上皮细胞表面补体片段C3a受体（C3aR）的激活对于单核趋化蛋白-1（CCL2)与受激活调节正常T细胞表达和分泌因子（CCL5)表达产生的影响。 方法：体外培养人肾皮质近曲小管上皮细胞（HK-2)，以0、75、150、300、600umol/L浓度尿酸（UA）分别作用6、12、24及48小时，利用RT-PCR检测CCL2、CCL5、C3和C3aR的mRNA水平，选择最适的尿酸诱导浓度和诱导时间。在UA或脂多糖（LPS）诱导下，通过小干扰RNA(siRNA)转染下调HK-2细胞C3的表达、小分子C3aR阻断剂阻断C3a-C3aR作用、外源性C3a刺激促进C3aR激活三种方式抑制或激活C3aR的信号通路，并通过Q-PCR及ELISA法检测其对于CCL2、CCL5表达产生的影响。 结果：150umol/L UA干预HK-2细胞12h可同时上调C3与CCL2的mRNA转录水平；在该实验中，UA对于HK-2细胞CCL5mRNA的诱导作用较弱，使用RT-PCR法在各浓度及时间点下未检测出明显统计学差异，此后我们改用LPS对CCL5进行诱导。C3siRNA转染或C3aR阻断能够抑制被UA或LPS上调的CCL2及CCL5的表达。C3a单独干预HK-2细胞时显著上调了CCL5的表达，但对CCL2的表达几乎无诱导作用。而当C3a与UA或LPS进行联合干预时，其显著增强了UA或LPS对CCL2的诱导作用及UA对CCL5的诱导作用。 结论：HK-2细胞表面C3aR的激活为CCL2、CCL5的表达提供了重要的刺激信号，阻断C3a-C3aR相互作用能够显著抑制UA或LPS诱导的HK-2细胞CCL2及CCL5的表达。
[Abstract]:Aim: to investigate the effects of the activation of complement fragment C 3a receptor (C3aR) on the expression of monocyte chemoattractant protein-1 (CCL2) and normal T cell (T cell) and secretory factor (CCL5) in the epithelial cells of proximal convoluted tubule of human renal cortex. Methods: human renal cortical proximal tubule epithelial cells (HK-2) were cultured in vitro and treated with uric acid (UA) at concentration of 075150300600umolL for 24 hours and 48 hours respectively. The mRNA levels of CCL2,CCL5,C3 and C3aR were detected by RT-PCR, and the optimal concentration and time of uric acid induction were selected. Under the induction of UA or lipopolysaccharide (LPS), the expression of C3 in HK-2 cells was down-regulated by small interfering RNA (siRNA), the effect of C3a-C3aR was blocked by small molecular C3aR blockers, and exogenous C3a stimulated C3aR activation to inhibit or activate C3aR signaling pathway. The expression of CCL2,CCL5 was detected by Q-PCR and ELISA. Results the mRNA transcription level of C _ 3 and CCL2 was up-regulated in HK-2 cells at 12 h after intervention by 1: 150umoll / L UA, and the induction of CCL5mRNA in HK-2 cells was weakly induced by CCL2. There was no significant difference between them by RT-PCR method at different concentrations and at different time points. Then we used LPS to induce. C3siRNA transfection or C3aR blockade could inhibit the expression of CCL2 and CCL5 up-regulated by UA or LPS. C3a significantly upregulated CCL5 expression in HK-2 cells, but had little effect on CCL2 expression. When C3a combined with UA or LPS, C3a significantly enhanced the induction of CCL2 by UA or LPS and CCL5 by UA. Conclusion the activation of C3aR on the cell surface provides an important stimulating signal for the expression of CCL2,CCL5. Blocking the C3a-C3aR interaction can significantly inhibit the expression of CCL2 and CCL5 in HK-2 cells induced by UA or LPS.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.6
，
本文编号：2247189
[Abstract]:Aim: to investigate the effects of the activation of complement fragment C 3a receptor (C3aR) on the expression of monocyte chemoattractant protein-1 (CCL2) and normal T cell (T cell) and secretory factor (CCL5) in the epithelial cells of proximal convoluted tubule of human renal cortex. Methods: human renal cortical proximal tubule epithelial cells (HK-2) were cultured in vitro and treated with uric acid (UA) at concentration of 075150300600umolL for 24 hours and 48 hours respectively. The mRNA levels of CCL2,CCL5,C3 and C3aR were detected by RT-PCR, and the optimal concentration and time of uric acid induction were selected. Under the induction of UA or lipopolysaccharide (LPS), the expression of C3 in HK-2 cells was down-regulated by small interfering RNA (siRNA), the effect of C3a-C3aR was blocked by small molecular C3aR blockers, and exogenous C3a stimulated C3aR activation to inhibit or activate C3aR signaling pathway. The expression of CCL2,CCL5 was detected by Q-PCR and ELISA. Results the mRNA transcription level of C _ 3 and CCL2 was up-regulated in HK-2 cells at 12 h after intervention by 1: 150umoll / L UA, and the induction of CCL5mRNA in HK-2 cells was weakly induced by CCL2. There was no significant difference between them by RT-PCR method at different concentrations and at different time points. Then we used LPS to induce. C3siRNA transfection or C3aR blockade could inhibit the expression of CCL2 and CCL5 up-regulated by UA or LPS. C3a significantly upregulated CCL5 expression in HK-2 cells, but had little effect on CCL2 expression. When C3a combined with UA or LPS, C3a significantly enhanced the induction of CCL2 by UA or LPS and CCL5 by UA. Conclusion the activation of C3aR on the cell surface provides an important stimulating signal for the expression of CCL2,CCL5. Blocking the C3a-C3aR interaction can significantly inhibit the expression of CCL2 and CCL5 in HK-2 cells induced by UA or LPS.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.6
，
本文编号：2247189
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