大鼠肝移植缺血再灌注后Kupffer细胞Toll样受体4及其伴侣分子MD-2的表达

Expression of Toll like receptor 4 and MD-2 gene and protein in Kupffer cellsfollowing ischemia-reperfusion injury in rat liver transplant

目的 :研究大鼠肝移植缺血再灌注后Kupffer细胞Toll样受体 4 (TLR4 )及MD - 2的表达 ,并探讨其在再灌注损伤中的作用。方法 :分离培养大鼠肝移植缺血再灌注后 0 (对照组 )、2、12、2 4h(再灌注组 )的Kupffer细胞 ,检测TLR4 /MD - 2mRNA、蛋白以及上清TNF -α的表达 ,并在培养液中加入抗TLR4抗体 (抗TLR4组 ) ,观察TLR4抗体对TNF -α分泌的影响。结果 :再灌注后Kupffer细胞TLR4 /MD - 2mRNA、蛋白以及TNF -α表达较对照组明显升高 (P <0 .0 1) ,应用抗TLR4抗体后 ,TNF -α量较再灌注组明显降低 (P <0 .0 1)。结论 :TLR4及其伴侣分子MD - 2在介导Kupffer细胞激活和肝移植缺血再灌注损伤中可能起重要作用。

Objective:To study the expression of Toll like receptor 4(TLR4) and MD-2 gene and protein in Kupffer cells and its role in ischemia-reperfusion injury (IRI) on rat liver graft.Methods:The Kupffer cells were isolated at 0(control group),2,12,24h (IR group)following IR.The mRNA and protein expressions of TLR4 and MD-2 were detected by RT-PCR and flow cytometry (FCM),respectively.The TNF-α level in supernatant was measured by ELISA.Then the isolated KCs were incubated with anti-TLR4 polyclonal antibody (anti-TLR4 group),and the TNF-α level were measured again.Results: The mRNA and protein expressions of TLR4/MD-2 and the level of TNF-α in IR group increased significantly following IR compared with those in control group( P <0.01).The TNF-α production was greatly inhibted after anti-TLR4 antibody treatment (compared with IR group, P <0.01).Conclusion:TLR4 and its partner molecule MD-2 may take an important part in the Kupffer cell activation and IRI.