發(fā)布時(shí)間：2018-09-18 07:19

【摘要】：目的：探討人腎皮質(zhì)近曲小管上皮細(xì)胞表面補(bǔ)體片段C3a受體（C3aR）的激活對(duì)于單核趨化蛋白-1（CCL2)與受激活調(diào)節(jié)正常T細(xì)胞表達(dá)和分泌因子（CCL5)表達(dá)產(chǎn)生的影響。 方法：體外培養(yǎng)人腎皮質(zhì)近曲小管上皮細(xì)胞（HK-2)，以0、75、150、300、600umol/L濃度尿酸（UA）分別作用6、12、24及48小時(shí)，利用RT-PCR檢測(cè)CCL2、CCL5、C3和C3aR的mRNA水平，選擇最適的尿酸誘導(dǎo)濃度和誘導(dǎo)時(shí)間。在UA或脂多糖（LPS）誘導(dǎo)下，通過(guò)小干擾RNA(siRNA)轉(zhuǎn)染下調(diào)HK-2細(xì)胞C3的表達(dá)、小分子C3aR阻斷劑阻斷C3a-C3aR作用、外源性C3a刺激促進(jìn)C3aR激活三種方式抑制或激活C3aR的信號(hào)通路，并通過(guò)Q-PCR及ELISA法檢測(cè)其對(duì)于CCL2、CCL5表達(dá)產(chǎn)生的影響。 結(jié)果：150umol/L UA干預(yù)HK-2細(xì)胞12h可同時(shí)上調(diào)C3與CCL2的mRNA轉(zhuǎn)錄水平；在該實(shí)驗(yàn)中，UA對(duì)于HK-2細(xì)胞CCL5mRNA的誘導(dǎo)作用較弱，使用RT-PCR法在各濃度及時(shí)間點(diǎn)下未檢測(cè)出明顯統(tǒng)計(jì)學(xué)差異，此后我們改用LPS對(duì)CCL5進(jìn)行誘導(dǎo)。C3siRNA轉(zhuǎn)染或C3aR阻斷能夠抑制被UA或LPS上調(diào)的CCL2及CCL5的表達(dá)。C3a單獨(dú)干預(yù)HK-2細(xì)胞時(shí)顯著上調(diào)了CCL5的表達(dá)，但對(duì)CCL2的表達(dá)幾乎無(wú)誘導(dǎo)作用。而當(dāng)C3a與UA或LPS進(jìn)行聯(lián)合干預(yù)時(shí)，其顯著增強(qiáng)了UA或LPS對(duì)CCL2的誘導(dǎo)作用及UA對(duì)CCL5的誘導(dǎo)作用。 結(jié)論：HK-2細(xì)胞表面C3aR的激活為CCL2、CCL5的表達(dá)提供了重要的刺激信號(hào)，阻斷C3a-C3aR相互作用能夠顯著抑制UA或LPS誘導(dǎo)的HK-2細(xì)胞CCL2及CCL5的表達(dá)。
[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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R692.6
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本文編號(hào)：2247189