摘要
为了建立奶牛骨髓源巨噬细胞的分离培养与鉴定方法,选用3种不同的培养基(RPMI-1640、DMEM、DMEM/F12)分别添加20%胎牛血清(FBS)、2.4%青-链霉素、1.2%谷氨酰胺(Gln)、M-CSF(20μg/L)将奶牛骨髓中提取出的单核细胞诱导为巨噬细胞,再通过加入脂多糖(Lipolyaccharide,LPS)将诱导出的M0型巨噬细胞极化为M1型巨噬细胞,光学显微镜在第1、4、7天观察巨噬细胞形态,比较3种培养基对分化巨噬细胞的差异。同时探究了前列腺素D_(2)(Prostaglandin D_(2),PGD_(2))-DP_(2)受体途径对大肠杆菌(Escherichia coli)诱导细胞因子(IL-6、TNF-α)分泌及巨噬细胞吞噬功能的影响。结果显示,RPMI-1640培养基中培养的细胞形态变化最为明显,且数量较多。并能够检测到大量的单核巨噬细胞特征性标志物(M0标志物:CD11b、CD14;M1标志物:CD11b、CD80)的表达,M0和M1型巨噬细胞纯度分别为79.9%和93.5%。与空白对照组相比,E.coli感染组COX-2和H-PGDS基因表达显著升高;PGD_(2)分泌量也显著上升(P<0.0001)。DP_(2)受体抑制剂(CAY10471、CAY10595)能够显著抑制E.coli诱导促炎性细胞因子(IL-6、TNF-α)的分泌和显著增强巨噬细胞对E.coli的杀伤作用。结果表明,诱导的细胞具有巨噬细胞特有的形态学特征和免疫表型;E.coli可诱导巨噬细胞PGD_(2)的产生,PGD_(2)-DP_(2)途径对E.coli感染的巨噬细胞细胞因子的分泌具有一定的调控作用。
In order to establish the isolation,culture and identification method of cow bone marrow-derived macrophages,three different media(RPMI-1640,DMEM,DMEM/F12)were added with 20%fetal bovine serum(FBS),2.4%chlorine-streptomycin,1.2%glutamine(Gln),and M-CSF(20 ng/mL),respectively,to induce the monocytes extracted from the bone marrow of dairy cows to become macrophages.The induced M0 macrophages were polarized into M1-type macrophages by adding lipopolysaccharide(LPS).The morphology of macrophages was observed by optical microscope at day 1,4 and 7,and the differences of differentiated macrophages between the three media were compared.The effects of prostaglandin D_(2)(PGD_(2))-DP_(2)receptor pathway on the secretion of cytokines(IL-6,TNF-α)induced by Escherichia coli and phagocytosis of macrophages were also investigated.The results showed that the morphological changes of cells cultured in the medium of RPMI-1640were the most obvious and the number was large.A large number of characteristic markers of mononuclear macrophages were detected(M0 markers:CD11b,CD14;M1markers:CD11b,CD80)expression,M0and M1 macrophage purity were 79.9%and 93.5%,respectively.COX-2and H-PGDS gene expressions were significantly increased in E.coli group compared with the blank control group.The secretion of PGD_(2)also increased significantly(P<0.0001).DP_(2)receptor inhibitors(CAY10471,CAY10595)could significantly inhibit the secretion of E.coli induced pro-inflammatory cytokines(IL-6,TNF-α)and significantly enhance the killing effect of macrophages on E.coli.The above results showed that the induced cells had the characteristic morphology and immunophenotype of macrophages.E.coli can induce the production of PGD_(2)in macrophages,and the PGD_(2)-DP_(2)pathway regulates the secretion of cytokines in E.coli infected macrophages.
作者
王钰
杨效林
郭莉莉
龚鹏飞
吴敬泽
毛伟
张双翼
刘博
曹金山
WANG Yu;YANG Xiaolin;GUO Lili;GONG Pengfei;WU Jingze;MAO Wei;ZHANG Shuangyi;LIU Bo;CAO Jinshan(College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China;Key Laboratory of Clinical Diagnosis and Treatment of Animal Diseases,Ministry of Agriculture and Rural Affairs,Hohhot 010018,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2024年第8期1674-1681,共8页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(32160851)
内蒙古自治区科技计划资助项目(2020GG0042)。
关键词
奶牛
骨髓源巨噬细胞
分离培养鉴定
PGD2
cow
bone marrow derived macrophages
isolation culture identification
PGD_(2)
