摘要
旨在探讨体外分离培养牛肺泡上皮细胞(bovine alveolar epithelial cells,BAECs)的方法及Wnt5a对牛结核分枝杆菌卡介苗(bacille Calmette-Guérin,BCG)感染BAECs细胞自噬的调控机制。试验选用酶联合消化法和机械刮刷法分离细胞,差速贴壁法纯化BAECs,免疫荧光染色检测上皮细胞标志物角蛋白14(cytokeratin 14,CK14)和角蛋白5(cytokeratin 5,CK5)的表达;BCG感染BAECs,并用Box-5抑制Wnt5a的表达,Western blot和免疫荧光染色检测自噬相关蛋白及非经典Wnt信号通路相关蛋白的表达。结果表明,采用酶联合消化法和机械刮刷法能够成功分离纯度较高的BAECs,细胞经CK14和CK5鉴定为阳性;BCG感染BAECs促进Wnt5a表达,增加细胞自噬,Box-5预处理下调BCG诱导的细胞自噬相关蛋白LC3II、P62、Atg7及Atg5的表达,且抑制非经典Wnt/Ca^(2+)信号通路相关蛋白Wnt5a、CaMKII及NFAT的表达。综上,试验成功建立BAECs分离培养方法,BCG感染增加BAECs内Wnt5a表达和细胞自噬,抑制Wnt5a下调BCG诱导的BAECs细胞自噬,且Wnt5a是通过非经典Wnt/Ca^(2+)信号通路调控BCG诱导的BAECs细胞自噬。
The aim of this study was to investigate the method of in vitro isolation and culture of bovine alveolar epithelial cells(BAECs) and the regulatory mechanism of Wnt5 a on the autophagy of BAECs infected with Mycobacterium tuberculosis bacille Calmette-Guérin(BCG).BAECs were isolated by enzyme combined digestion method and mechanical scraping method,and purified by differential adhesion method.The expression of epithelial cell markers CK14 and CK5 were detected by immunofluorescence staining.BAECs were infected with BCG and the expression of Wnt5 a was inhibited by Box-5.The expression of autophagy-related proteins and nonclassical Wnt signaling pathway related proteins were detected by Western blot and immunofluorescence staining.The results showed that BAECs were successfully isolated with high purity by enzyme combined digestion and mechanical scraping.Isolated cells were identified as positive by CK14 and CK5.BCG infection with BAECs promoted the expression of Wnt5 a and increased autophagy.Box-5 pretreatment down-regulated the expression of BCG-induced autophagy-related proteins LC3 II,P62,Atg7 and Atg5,and inhibited the expression of nonclassical Wnt/Ca^(2+) signaling pathway related proteins Wnt5 a,CaMKII and NFAT.In conclusion,the isolation and culture method of BAECs was successfully established.BCG infection promoted the Wnt5 a expression and autophagy of BAECs,and the inhibition of Wnt5 a expression down-regulated the BCG-induced autophagy in BAECs.Moreover,Wnt5 a regulates the autophagy induced by BCG in BAECs through the nonclassical Wnt/Ca^(2+) signaling pathway.
作者
陈琪
杜长征
郑雪迪
马伯利
徐金瑞
杨易
CHEN Qi;DU Changzheng;ZHENG Xuedi;MA Boli;XU Jinrui;YANG Yi(Key Laboratory of Education for Conservation and Utilization of Special Biological Resources in the Western China,Ningxia University,Yinchuan 750021,China;College of Life Science,Ningxia University,Yinchuan 750021,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2022年第11期4071-4080,共10页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(31960712,31960700)。
