热射病大鼠枯否细胞功能变化及其机制研究

Changes in Secretion and Phagocytosis of Rat Kupffer Cells Under Heat-stroke and Its Mechanism

目的观察热射病(heat stroke,HS)大鼠枯否细胞(kupffer cells,KCs)吞噬及分泌功能的变化并探讨其可能机制。方法用动物体温维持仪建HS大鼠动物模型。对照组及HS组大鼠肝脏苏木精-伊红(hematoxylin-eosin,HE)染色,观察KCs吞噬印度墨水的情况。留取各组大鼠实验前后外周血,鲎试剂盒检测内毒素浓度,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)试剂盒检测巨噬细胞炎症蛋白1α(macrophage inflammatory protein-1 alpha,MIP-1α)、肿瘤坏死因子α(tumor necrosis factor-alpha,TNF-α)、白介素1β(interleukin-1 beta,IL-1β)及干扰素γ(interferongamma,INF-γ)浓度。制备石蜡切片,免疫荧光技术检测大鼠KCs内磷酸化c-Jun氨基端激酶(phosphorylation c-Jun Nterminal kinase,p-JNK)及MIP-1α的表达。用细胞裂解液匀浆提取肝组织总蛋白,蛋白免疫印迹(Western blot)检测各组大鼠KCs内p-JNK及MIP-1α蛋白的表达。结果大鼠肝脏HE染色显示HS组与对照组相比,KCs吞噬印度墨水的能力明显下降(P<0.05)。HS组大鼠外周血内毒素浓度明显高于对照组(P<0.05);MIP-1α、TNF-α、IL-1β及INF-γ浓度明显高于对照组(P<0.05)。大鼠肝脏免疫荧光结果显示p-JNK和MIP-1α主要表达在KCs,与对照组相比,HS组KCs内p-JNK和MIP-1α的表达明显增多,HS组大鼠肝脏p-JNK和MIP-1α蛋白表达均明显上调(P<0.05)。结论 HS组大鼠KCs吞噬功能减弱,分泌TNF-α、IL-1β及INF-γ等炎症因子的能力增强,其机制可能与c-Jun氨基端基酶信号通路激活及MIP-1α表达增多有关。

Objective To investigate the secretion and phagocytosis changes of rat kupffer cells （KCs） under heat stroke （HS） and its mechanism. Methods The Wistar rats placed on body temperature maintenance were randomly as- signed to normal control（NC）group and HS group. The rat livers of NC and HS groups were all dyed by hematoxylin-eo- sin （HE） staining, and the cytophagic situation of Indian ink in KCs were observed. All rat peripheral blood samples were collected before and after the experiment, endotoxin concentrations were detected by limulus test kit, and concerntrations of macrophage inflammatory protein-1 alpha （MIP-1β）, tumor necrosis factor-alpha （TNF-α）, interleukin-1 beta （IL- 113）, interferon-gamma （INF-γ） were all tested by enzyme linked immunosorbent assay （ELISA）. The paraffin sections were prepared, expressions of phosphorylation c-Jun N-terminal kinase （p-JNK） and MIP-1β in rat KCs were assessed by using immunofluorescence technique. The cell lysis buffer and Western-blot were used to extract total protein of liver tis- sues and detect the expressions of p-JNK as well as MIP-la in rat KCs, respectively. Results HE staining results of rat liver showed phagocytosis with Indian ink of KCs in HS group was predominantly weaker than that of NC group （P〈0. 05）. The concentrations of endotoxin in rat peripheral blood, MIP-la, TNF-α, IL-1βand INF-γ in HS group were remarkably more than these of NC group （P〈0. 05）. The expressions of p-JNK and MIP-la were mainly expressed in KCs and liver by immunofluorescence. Compared with NC group, the expressions of p-JNK and MIP-1α in KCs of HSgroup obviously up-regulated （P〈0. 05）. Conclusion The phagocytosis of HS rat decreses and secretory functions increase in inflammatory factors, such as TNF- α, IL-1β, INF-γ, etc. Its mechanism may relate to acti- vate JNK signal path and grow up with MIP-la expres-sions.