左氏无绿藻对奶牛乳腺上皮细胞侵染凋亡的机制研究

Bovine mastitis cases caused by Prototheca zopfii have been reported in 21 countries all over the world, which were considered to be pathogenic and refractory. The morbidity of this diesease is becoming popular and has been focused by researchers and veterinarian for bovine mastitis. Currently, Prototheca zopfii strains firstly isolated from Chinese milk samples of cows suffering from mastitis through Beijing, Tianjing and Shandong dairy farms by our group and the results have been published on SCI journals. However, the mechanism of this kind of mastitis has not been elucidated, particularly, the molecular characteristics of epithelial cell line from bovine mammary gland adhering, invading and apoptosis by Prototheca zopfii are not clear until now. The objective of this program is therefore, to observe Prototheca zopfii adhered BMEC by scanning electronic microscopy and transmission electronic microscopy, and apoptosis of BMEC by hoechst33258 staining, to analyze Prototheca zopfii adhered and invaded BMEC cytokines Fas, caspases, p53, bcl-2、bax、p300/CBP and ROS by qPCR, western blot and flow cytometry, to study the effect of Prototheca zopfii adhered and invaded BMEC cytokines included IL-1、IL-6、IL-12、TNF、IFN, and immune responses TLR2 and TLR4 by Real-time PCR, western blot and ELISA. The program finally demonstrated the molecular characteristics of epithelial cell line from bovine mammary gland adhering, invading and apoptosis by Prototheca zopfii.

已有21个国家和地区报道左氏无绿藻导致奶牛发生乳房炎，具有爆发性和高致病性，属于人畜共患病。其发病率和传播率正在逐渐增高和扩大，已经引起业界的广泛关注。本课题组已经在我国北京、天津和山东奶牛乳房炎的乳汁中分离鉴定出左氏无绿藻。但是它对奶牛乳腺上皮细胞侵染凋亡的机制还不清楚。本研究旨在通过我们分离的左氏无绿藻体外感染奶牛乳腺上皮细胞，利用透射电镜观察左氏无绿藻对奶牛乳腺上皮细胞的侵染，Hoechst33258染色法检测上皮细胞的凋亡过程，分析左氏无绿藻介导奶牛乳腺上皮细胞凋亡时死亡受体Fas，线粒体凋亡酶激活因子Caspases，MAPK通路中p53、bcl-2、bax、p300/CBP和ROS，炎性细胞因子IL-1、IL-6、IL-12、TNF、IFN，模式识别受体TLR2和TLR4及其NF-κB信号传递途径的变化，以此阐明左氏无绿藻对奶牛乳腺上皮细胞侵染凋亡的机制。

已有24个国家和地区报道人畜共患的牛无绿藻（左氏无绿藻II）导致奶牛发生乳房炎，具有爆发性和高致病性。其发病率和传播率正在逐渐增大，引起业界的广泛关注。本课题组在我国北京、天津和山东采集620份奶牛乳房炎乳汁，分离鉴定出84株牛无绿藻，占13.5%。它对奶牛乳腺上皮细胞（bMECs）和小鼠乳腺的致病机制以及抗菌肽-蛋白酶抑制素对乳腺炎模型的先天性免疫保护机制还不清楚。本研究旨在通过细胞培养、荧光定量PCR，Western blotting，共聚焦激光扫描显微镜，流式细胞术、扫描电镜，透射电镜和末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记等技术研究牛无绿藻的致病机制。结果表明：牛无绿藻通过NF-κB信号途径部分调节bMECs先天性免疫应答和炎症反应，它对bMECs的粘附力高于西弗无绿藻，牛无绿藻的调节能力显著高于西弗无绿藻，从而引起严重的乳腺组织炎症。病理形态学、氧化应激和凋亡的结果表明，牛无绿藻通过氧化和抗氧化防御的失衡以及细胞内ROS的产生而诱导bMECs凋亡和氧化应激。超微结构显示，牛无绿藻具有侵袭bMECs的能力，对乳腺组织造成显著的损伤，还导致细胞凋亡。通过建立牛无绿藻小鼠乳腺炎模型，研究其在小鼠乳腺上皮细胞和巨噬细胞炎症和凋亡中的作用，结果显示牛无绿藻在乳腺组织中大量复制，引起乳腺组织严重的炎症，巨噬细胞和中性粒细胞浸润，促炎基因上调，上皮细胞凋亡。利用该模型，研究抗菌肽-蛋白酶抑制素对乳腺上皮细胞的先天性免疫保护作用，可知蛋白酶抑制素参与牛无绿藻的发病过程，蛋白酶抑制素在多肽家族中具有独特的作用。内源性蛋白酶抑制素是乳腺组织中天然防御牛无绿藻的关键因素，人源蛋白酶抑制素（LL-37）具有杀灭无绿藻和免疫调节作用。本研究率先揭示牛无绿藻是一种引起乳腺炎的病原体，可引发严重的炎症和线粒体凋亡，蛋白酶抑制素具有保护作用。截止目前，本课题已发表SCI论文6篇，累积IF=19.557，被引25频次。培养硕士和博士生3名。