摘要
为了探究大肠杆菌(E.coli)对藏羊子宫内膜上皮细胞(EECs)相关凋亡因子表达的影响,采用酶消化法培养EECs,用不同感染复数(MOI)进行E.coli感染试验,通过流式细胞术(Flow CytoMetry,FCM)检测EECs的凋亡率,用实时荧光定量PCR(qRT-PCR)及免疫印迹法(Western Blot)检测Caspase-3、Bcl-2和Bax等凋亡因子RNA和蛋白的表达水平。结果显示,在培养液中加入50 ng/mL表皮生长因子(EGF)能成功培养出EECs,其纯度达到98%以上;E.coli感染试验中,当MOI为1∶1时,Caspase-3和Bax在EECs的表达量与对照组相比变化不明显,而Bcl-2的表达量显著升高;当MOI为20∶1时,Bax的表达量显著升高;当MOI为50∶1时,Caspase-3的表达量显著升高,而Bcl-2的表达量与对照组相比变化不明显。结果表明,酶消化法可成功培养EECs;不同MOI的E.coli感染后,EECs中Caspase-3、Bcl-2和Bax等凋亡因子的表达量较空白组有不同程度的升高,这些凋亡因子可作为识别子宫内膜炎病理变化程度的指标。
In order to investigateeffectof E.coli on the expression of apoptosis-related factors in Tibetan sheep endometrial epithelial cells(EECs),EECs were cultured in vitro by enzymatic digestion and infected with E.coli at different multiplicity of infections(MOI),the apoptosis rate of EECs was detected by flow cytometry(FCM),and the expressions of apoptotic factors such as Caspase-3,Bcl-2,and Bax were detected by quantitative real-time PCR(qRT-PCR)and Western blot respectively.The results showed that EECs were successfully cultured in DMEM with 50 ng/mL EGF,and the purity rate of the cells was 98%;EECs were infected with E.coli for 3 hours in vitro at different MOIs.The expressions of Caspase-3 and Bax genes and proteins did not change significantly compared with the control when MOI was 1∶1,while those of Bcl-2 increased dramatically;when MOI was 20∶1,the expression of Bax increased;when MOI was 50∶1,the expression of Caspase-3 increased significantly,while that of Bcl-2 remained at the same level.The results showed that the expression levels of Caspase-3,Bcl-2,and Bax in EECs cultured in vitro were significantly higher than those of the control after being infected with E.coli at different MOI,and these factors can be taken as indicators to identify the pathological changes of endometritis in Tibetan sheep.
作者
王立斌
张兴云
王萌
潘阳阳
胡学权
韩金辉
马进彪
马文斌
徐庚全
樊江峰
余四九
WANG Libin;ZHANG Xingyun;WANG Meng;PAN Yangyang;HU Xuequan;HAN Jinhui;MA Jinbiao;MA Wenbin;XU Gengquan;FAN Jiangfeng;YU Sijiu(College of Veterinary Medicine,Gansu Agricultural University,Technology and Research Center of Gansu Province for Embryo Engineering of Bovine and Sheep&Goat,Lanzhou 730070,China)
出处
《西北农业学报》
CAS
CSCD
北大核心
2021年第4期490-498,共9页
Acta Agriculturae Boreali-occidentalis Sinica
基金
国家重点研发计划(2017YFD0502200)
甘肃省农业科技创新项目(GNSW-2015-19)。
