Expression of toll-like receptor 4 and MD-2 gene and protein in Kupffer cells after ischemia-reperfusion in rat liver graft.
Expression of toll-like receptor 4 and MD-2 gene and protein in Kupffer cells after ischemia-reperfusion in rat liver graft.
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DOI：
10.3748/wjg.v10.i19.2890
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发表时间：
2004-10
影响因子：
4.3
通讯作者：
Yong Peng;J. Gong;Chang-An Liu;Xu-hong Li;L. Gan;Shou-bai Li
中科院分区：
文献类型：
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作者：
Yong Peng;J. Gong;Chang-An Liu;Xu-hong Li;L. Gan;Shou-bai Li
目的研究大鼠移植肝缺血再灌注损伤后枯否细胞（KCs）Toll样受体4（TLR 4）和MD-2基因及蛋白的表达变化，探讨其在移植肝缺血再灌注损伤中的作用。方法取大鼠移植肝，分别于缺血再灌注（IR）后0（对照组）、2、12、24 h（IR组）分离KCs。RT-PCR检测TLR 4和MD-2 mRNA表达，流式细胞术（FCM）检测TLR 4/MD-2蛋白表达，ELISA检测培养上清中肿瘤坏死因子-α（TNF-α）水平。然后将分离的KCs与抗TLR 4多克隆抗体（抗TLR 4组）孵育，再次测量TNF-α水平。结果IR组TLR 4/MD-2 mRNA和蛋白表达及TNF-α水平在IR后2 h即显著升高，12 h达高峰，24 h略有下降，但仍显著高于对照组（P<0.01）。抗TLR 4抗体治疗后，上述因子的表达较IR组明显降低（P<0.01）。结论IR后脂多糖（LPS）可上调KCs TLR 4/MD-2基因和蛋白表达，并合成细胞因子TNF-α。抗TLR 4抗体可抑制LPS诱导的TNF-α的产生。TLR 4及其伴侣分子MD-2可能在库普弗细胞活化和IR损伤中起重要作用。
AIM To investigate the expression of toll-like receptor 4 (TLR4) and MD-2 gene and protein in Kupffer cells (KCs) and their role in ischemia-reperfusion (IR) injury of rat liver graft. METHODS KCs were isolated at 0 (control group), 2, 12, 24 h (IR group) following IR in rat liver graft. mRNA expression of TLR4 and MD-2 was detected by RT-PCR analysis, protein expression of TLR4/MD-2 was detected by flow cytometric (FCM) analysis, and tumor necrosis factor-alpha (TNF-alpha) level in supernatant was measured by ELISA. Then isolated KCs were incubated with anti-TLR4 polyclonal antibody (anti-TLR4 group), and TNF-alpha level was measured again. RESULTS The mRNA and protein expression of TLR4/MD-2 and the level of TNF-alpha in IR group increased significantly at 2 h following IR, and reached the maximum at 12 h, and slightly decreased at 24 h, but were still significantly higher than those in the control group (P<0.01). The expression of these factors was markedly decreased after anti-TLR4 antibody treatment as compared with the IR group (P<0.01). CONCLUSION Lipopolysaccharide (LPS) following IR can up-regulate TLR4/MD-2 gene and protein expression in KCs, and synthesize cytokine TNF-alpha. Anti TLR4 antibody can inhibit the production of TNF-alpha induced by LPS. TLR4 and its partner molecule MD-2 may play an important role in Kupffer cell activation and IR injury.