肝实质细胞抑制细菌增殖作用及机制研究

Inhibitory effect and underlying mechanism of hepatocytes on bacterial proliferation

目的探讨肝细胞通过直接吞噬和抗菌蛋白介导抑制细菌增殖的作用及其机制。方法利用正常小鼠FL83B肝细胞系建立大肠杆菌感染模型,以小鼠Kupffer细胞系为对照,观察肝细胞对大肠杆菌的增殖影响;建立细菌-细胞和(或)细胞上清共培养体系,通过Kupffer/FL83B细胞及其6、12、24 h的上清与大肠杆菌的共培养,检测大肠杆菌的致死情况,明确肝细胞直接抑菌效应;通过吞噬功能检测肝细胞的吞噬能力,qRT-PCR、Western blot检测相关基因的表达。结果将大肠杆菌与细胞共孵育,发现FL83B细胞与Kupffer细胞类似,可直接抑制大肠杆菌的增殖(P<0.05),且肝细胞自身保持高存活率;通过吞噬功能检测,明确了FL83B细胞可直接吞噬细菌颗粒;与对照组相比,感染后肝细胞内吞噬相关基因Fcgr1、Marco、Cdc42、RhoB mRNA上调,抗原递呈相关基因β2M mRNA上调,NF-κB、MAPK、STAT3通路活化,且介导抗菌作用的相关蛋白iNOS、NOX2、RhoB表达上调(P<0.05)。结论小鼠FL83B肝细胞可直接通过吞噬作用和抗菌相关蛋白介导抑制细菌增殖。

Objective To explore the role and mechanism of hepatocytes in inhibiting bacterial proliferation by phagocytizing bacteria directly and mediating the expression of antibacterial proteins indirectly.Methods Cellular model of Escherichia coli(E.coli)infection was established in normal murine hepatocyte line FL83B and mouse Kupffer cells to determine the effects of the cells on the proliferation of bacteria.After the bacteria were co-cultured with Kupffer/FL83B cells and the 6-,12-,and 24-hour supernatants,respectively,the lethality of E.coli was determined for the direct bacteriostatic effect of the hepatocytes.The phagocytizing ability of FL83B cells were measured with phagocytosis reagent kit.qRT-PCR and Western blotting were employed to detect the expression of related molecules at mRNA and protein levels.Results When the hepatocyte were incubated with E.coli,FL83B cells,as Kupffer cells,could directly inhibit the proliferation of the bacteria(P<0.05)and maintained a high survival rate at same time.Phagocytic function test showed that FL83B cells could directly phagocytize bacterial particles.Compared with the FL83B cells without E.coli infection,the mRNA levels of phagocytosis-related genes Fcgr1,Marco,Cdc42,and RhoB were increased,that of antigen presentation related geneβ2M was also upregulated,the NF-κB,MAPK,and STAT3 signaling pathways were activated,and the protein levels of antibacterial-related proteins iNOS,NOX2,and RhoB were also enhanced in the cells after infection(all P<0.05).Conclusion Mouse FL83B hepatocytes possess the ability to inhibit bacteria through direct phagocytosis and indirect upregulation of antibacterial-related proteins.