LPS诱导条件下猪小肠上皮细胞TLR4及其信号通路基因表达变化分析

Analysis of Differential Expression of TLR4 and TLR4 Signaling Pathway Genes under Lipopolysaccharide-induced Pig Intestinal Epithelial Cells

本试验通过0.1和1μg·mL-1浓度的LPS诱导处理猪小肠上皮细胞(IPEC-J2),分别在2、4、6h时利用实时荧光定量PCR方法检测TLR4及其信号通路相关基因(CD14、MyD88、TNF-α、IL-1β和IFN-α)mRNA水平相对表达量,初步探讨猪小肠上皮细胞受到产肠毒素大肠杆菌侵扰发生炎症反应的相关分子反应机理。结果发现,两种浓度的LPS均使得所检测的TLR4及其信号通路相关基因表达量上调,诱导后4~6h的表达量急速上升,且高浓度的LPS诱导处理后各基因表达量上调倍数明显高于低浓度LPS诱导时各基因表达量上调倍数,高浓度的LPS对机体肠道的刺激引起了更为强烈的免疫反应,使正常机体更快地产生炎症反应。由此推测,大肠杆菌侵染猪肠道后将释放LPS,TLR4作为LPS的受体,受LPS诱导其表达量上调,进而引起TLR4信号途径的信号传递,传递过程中由于MyD88的依赖机制,MyD88表达量上调相对稳定,再经过级联免疫放大效应,大量的促炎细胞因子释放,导致炎症及腹泻水肿病的产生。

In this study,we exposed pig intestinal epithelial cells(IPEC-J2)to 0.1 and 1μg·mL-1 LPS for 2,4and 6h,respectively.Then,we estimated the relative mRNA expression of TLR4 and TLR4signaling pathway-related genes(CD14,MyD88,TNF-α,IL-1β,IFNα)using qPCR,which preliminary revealed the mechanism of the molecules related to inflammatory reactions in pig intestinal epithelial cells induced by enterotoxigenic Escherichia coli.Both concentrations of LPS upregulated the expression of TLR4 and its signaling pathway-related genes,and the expression level of all genes sharply increased from 4to 6h.The fold change of mRNA expression induced by 1.0μg·mL-1 LPS was significantly higher than that induced by 0.1μg·mL-1 LPS.The former stimulated the intestinal tract to produce stronger immune responses and more rapid development of inflammatory reactions.Above results suggested that LPS was released in the pigintestinal tract with E.coli infection,and upregulated the LPS receptor TLR4,leading to activation of the TLR4 signaling pathway.Given the dependency on myeloid differentiation factor 88(MyD88)during signaling,stable upregulation of MyD88 and the cytokine cascade results in the release of large amounts of proinflammatory cytokines,causing inflammatory reactions,diarrhea and edema disease.