摘要
试验旨在探索革兰氏阴性菌大肠杆菌(Escherichia coli,E.coli)及其表面分子脂多糖(LPS)诱导胰腺再生蛋白Ⅲγ(RegⅢγ)表达调控的机制。首先,用不同浓度灭活E.coli(10^(9)、10^(8)、10^(7)、10^(6)、10^(5)、10^(4)CFU/mL)和LPS(0.01、0.1、1、5、10、20、40、80μg/mL)诱导猪肠黏膜上皮细胞(IPEC-JⅡ),用MTT法测D490 nm值,检测E.coli和LPS对IPEC-JⅡ细胞活力的影响;其次,用不同浓度灭活E.coli(10^(7)、10^(6)、10^(5)CFU/mL)和LPS(0.01、0.1、1、5μg/mL)处理IPEC-JⅡ细胞24 h,用实时荧光定量PCR和Western blotting检测RegⅢγmRNA和蛋白的表达;最后,用1μg/mL LPS处理IPEC-JⅡ细胞24 h,用实时荧光定量PCR和Western blotting检测p65、p38、JNK、ERK mRNA和蛋白表达及磷酸化水平。结果显示,除0.01μg/mL LPS不抑制IPEC-JⅡ细胞活力外,其他浓度的灭活E.coli和LPS均可抑制IPEC-JⅡ细胞活力,且10^(9)、10^(8)CFU/mL E.coli和10、20、40、80μg/mL LPS组细胞活力极显著下降(P<0.01);与对照组相比,10^(7)、10^(6)和10^(5)CFU/mL E.coli均能诱导RegⅢγ表达增加,且10^(5)CFU/mL E.coli组RegⅢγmRNA表达量极显著高于对照组(P<0.01),蛋白表达量显著高于对照组(P<0.05);0.01、0.1、1和10μg/mL LPS均能诱导RegⅢγ表达增加,且0.1和1μg/mL LPS组RegⅢγmRNA表达量极显著高于对照组(P<0.01),RegⅢγ蛋白表达虽有增加趋势,但差异不显著(P>0.05);与对照组相比,1μg/mL LPS组p65、p38 mRNA表达量极显著增加(P<0.01),JNK、ERK mRNA表达量显著增加(P<0.05);同时,p38、JNK蛋白表达量和磷酸化水平均极显著增加(P<0.01),p65蛋白磷酸化水平显著增加(P<0.05),ERK蛋白和磷酸化水平均增加,但差异不显著(P>0.05)。以上结果表明,灭活E.coli和LPS均可诱导RegⅢγ表达,1μg/mL LPS可增加p65、p38和JNK蛋白的磷酸化水平。
This experiment was intended to explore the regulation mechanism of Gram-negative bacterium Escherichia coli(E.coli)and its surface molecules lipopolysaccharide(LPS)induced the pancreas regeneration proteinⅢgamma(RegⅢγ)expression.Firstly,different concentration E.coli(10^(9),10^(8),10^(7),10^(6),10^(5)and 10^(4)CFU/mL)and LPS(0.01,0.1,1,5,10,20,40 and 80μg/mL)were used to induce intestinal epithelia cells of porcine(IPEC-JⅡ),and D490 nmvalue was determined by MTT method to judge the effect of E.coli and LPS on IPEC-JⅡcell viability.Then,different concentration E.coli(10^(7),10^(6)and 10^(5)CFU/mL)and LPS(0.01,0.1,1 and 5μg/mL)were used to induce IPEC-JⅡfor 24 h,and Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression of RegⅢγ.Finally,1μg/mL LPS was used to treat IPEC-JⅡfor 24 h,and Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression and phosphorylation level of p65,p38,JNK and ERK.The results showed that except 0.01μg/mL LPS did not inhibit IPEC-JⅡcell viability,all the other concentration of E.coli and LPS inhibited IPEC-JⅡcell viability,moreover,the cell viability of 10^(9)and 10^(8)CFU/mL E.coli and 10,20,40,80μg/mL LPS group were extremely significantly decreased(P<0.01).Compared with control group,10^(7),10^(6)and 10^(5)CFU/mL E.coli could increase the expression of RegⅢγin IPEC-JⅡcells,and the RegⅢγmRNA expression of 10^(5)CFU/mL E.coli group was extremely significantly higher than that of control group(P<0.01),the quantity of protein expression was significantly higher than control group(P<0.05).0.01,0.1,1 and 10μg/mL LPS could increase the expression of RegⅢγin IPEC-JⅡcells,moreover,the RegⅢγmRNA expression of 0.1 and 1μg/mL LPS group was extremely significantly higher than that of control group(P<0.01),although RegⅢγprotein expression had increasing trend,but had no significant difference(P>0.05).Compared with control group,the mRNA expression of p65 and p38 of 1μg
作者
孟江海
向琼昊
宋云云
曹斌
陈韬
MENG Jianghai;XIANG Qionghao;SONG Yunyun;CAO Bin;CHEN Tao(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China;Hunan Veterinary Drug Engineering and Technology Research Center,Changsha 410128,China)
出处
《中国畜牧兽医》
CAS
北大核心
2021年第3期846-854,共9页
China Animal Husbandry & Veterinary Medicine
基金
湖南省教育厅科研项目重点项目(18A109)。
