胰腺再生蛋白Ⅲγ在猪小肠上皮细胞中的表达调控机理

Expression regulation mechanism of the pancreatic regenerative proteinⅢγin porcine intestinal epithelial cells

选取不同浓度(0、10^(4)、10^(5)、10^(6)、10^(7)、10^(8)、10^(9)cfu/mL)的灭活金黄色葡萄球菌(Staphylococcus aureus)和不同质量浓度(0.0、0.1、1.0、10.0μg/mL)的肽聚糖(PG)诱导猪小肠上皮细胞(IPEC–J2),通过q PCR和免疫印迹试验,检测RegⅢγmRNA和蛋白的表达水平,分析RegⅢγ及P65、P38、c–Jun氨基末端激酶(JNK)、细胞外调节蛋白激酶(ERK)等的表达变化,进一步探究RegⅢγ表达调控的分子机理。结果表明:灭活S.aureus和PG对IPEC–J2的细胞活力均有抑制作用;与不添加灭活S.aureus处理相比,添加灭活S.aureus可增加RegⅢγmRNA和蛋白的表达,且呈现剂量依赖性,当灭活S.aureus浓度达到10;cfu/mL时,RegⅢγmRNA和蛋白的表达水平极显著增加;同样PG也能诱导RegⅢγ蛋白的表达,与不添加PG的处理相比,添加1.0μg/mL PG时,RegⅢγmRNA和蛋白表达水平极显著升高;与不添加灭活S.aureus或PG的处理相比,添加10^(9)cfu/mL灭活S.aureus或1.0μg/mL PG能显著或极显著提高IPEC–J2的P65、P38、JNK、ERK等蛋白的表达水平,表明RegⅢγ在IPEC–J2中的表达可能是由髓样分化初级应答蛋白(MyD88)下游的核转录因子κB(NF–κB)和丝裂原活化蛋白激酶(MAPK)2条信号通路途径所调控。

A group of the concentrations of inactivated Staphylococcus aureus(0,10^(4),10^(5),10^(6),10^(7),10^(8),10^(9)cfu/mL)and of peptidoglycan(PG)(0.0,0.1,1.0,10.0μg/mL)were selected to induce the expression of regenerating proteinⅢγ(Reg IIIγ)in porcine intestinal epithelial cells(IPEC-J2).The levels of RegIIIγm RNA and protein were detected by q PCR and western blot assay,and the expression variations of RegⅢγ,P65,P38,C-Jun amino terminal kinase(JNK)and extracellular regulatory protein kinase(ERK)were analyzed to further explore the molecular mechanism of RegⅢγexpression regulation.The results showed that both inactivated S.aureus and PG had inhibitory effects on cellular viability of IPEC-J2.In comparison to the treatment without inactivated S.aureus,the mRNA and protein expression levels of RegⅢγwas higher in the groups of inactivated S.aureus treatment and showed a does-dependent trend,and the m RNA and protein expression levels were significantly increased when the concentration of inactivated S.aureus was 10;cfu/mL.Similarly,PG also induced the expression of RegⅢγ.Compared with the treatment without PG,the mRNA and protein expression levels of RegⅢγwere significantly higher when PG was 1.0μg/mL.The expression levels of P65,P38,JNK,and ERK in IPEC-J2 were significantly increased by adding 10^(9)cfu/mL inactivated S.aureus or 1.0μg/mL PG,compared with that without adding inactivated S.aureus or PG.These results suggested that the expression of RegⅢγin IPEC-J2 might be regulated by two signaling pathways:nuclear transcription factorκB(NF-κB)and mitogen-activated protein kinase(MAPK)downstream of the primary myeloid differentiation response protein MyD88.