ACE 2在奶牛乳腺中的表达定位及其抗炎性损伤作用的研究

doi:10.11843/j.issn.0366-6964.2017.03.020

摘要/Abstract

摘要：

本研究旨在通过体内外试验，明确ACE 2在奶牛乳腺中的表达定位，并对其在高精料饲喂致内源性奶牛乳房炎中的抗损伤作用进行初步探讨。体外试验分别选用牛乳腺组织样品和牛乳腺上皮细胞（Mac-T细胞），RT-PCR和Western blotting法检测ACE 2在奶牛乳腺组织中的mRNA和蛋白表达；免疫荧光法测定ACE 2蛋白在Mac-T细胞中的定位。体内试验，选用6头健康泌乳期荷斯坦奶牛，随机分为对照组和高精料饲喂组，饲喂20周后,活体采集乳腺组织及乳。测定乳中LPS含量、体细胞数及相关炎性指标；制作乳腺组织学切片；分析乳腺组织中ACE、AT 1、ACE 2、MasR mRNA表达水平, ELISA法检测Ang II、Ang1-7含量。体外试验结果表明，奶牛乳腺中ACE 2在基因和蛋白水平上均有表达，细胞学定位在细胞的胞浆和胞膜。体内试验结果显示，高精料长期饲喂，奶牛乳中体细胞数和LPS含量均明显升高（P<0.01）；乳中NAGase和AKP酶活力均显著高于正常对照组(P<0.05)，MPO活力两组之间无明显差异；乳腺组织学表现出一定炎性损伤变化；其中Ang1-7含量显著增高（P<0.05），Ang II的含量显著降低（P<0.05）；ACE、AT 1、ACE 2、MasR mRNA表达量均显著上升（（P<0.05）），ACE 2/ACE mRNA比值高于对照组。本研究首次证实奶牛乳腺组织中有ACE 2的存在。在高精料长期饲喂造成奶牛乳房炎性损伤时，乳腺组织中RAS处于激活状态，ACE/ACE 2比例失衡，以ACE 2-Ang1-7-MasR轴的活力处于优势状态，即ACE 2的抗损伤作用占优势。ACE 2可能通过降解AngII，产生Ang1-7发挥抗损伤作用, 对乳腺组织炎性损伤具有一定的保护作用。

Abstract:

The aim of this study is to gain a clear understanding of the expression distribution of ACE 2 through both in vivo and in vitro experiments and also to study ACE 2’s anti-injury effect in dairy cow mastitis induced by long-term high concentrate feeding. In vitro, mammary tissues samples from the lactating Holstein cows and Mac-T cells were used. The mRNA and protein expression of ACE 2 in bovine mammary gland was determined using RT-PCR and Western blotting. The location of ACE 2 protein in Mac-T cell was determined using immune fluorescence method. In vivo, 6 healthy lactating Holstein cows were divided into 2 groups: normal feeding group (40% concentrate, dry matter basis) and high concentrate feeding group (60% concentrate). After 20 weeks’ feeding, mammary biopsy samples and milk samples were collected. Milk LPS content, somatic cell number, as well as some inflammatory indicators including NAGase, AKP and MPO were tested. Histopathological test was also performed. The mRNA expression level of ACE 2, ACE and their receptors AT 1 and MasR in mammary tissues were analysed using RT-qPCR. The contents of AngII and Ang1-7 were also determined using ELISA.The results showed that both mRNA and protein expression of ACE 2 were found in dairy cow mammary gland, ACE 2 was located in the cytoplasm and on the membrane of the Mac-T cell. With a long-term high concentrate feeding, somatic cell number and LPS content in milk significantly increased (P<0.01); the activities of NAGase and AKP were significantly higher than those in the control group (P<0.05), MPO activity had no significant differences between the 2 groups; mammary gland tissues in the high concentrate group cows found inflammatory injury changes. The mRNA expression level of ACE, ACE 2, AT 1, MasR increased, and the mRNA ratio of ACE 2/ACE was higher than the control group. Ang1-7 content was significantly higher in breast tissue (P<0.05), AngII content was significantly lower ( P<0.05).Conclusion: It is the first time that the presence of ACE 2 in bovine mammary gland has been confirmed. When mammary inflammatory injuries are induced by long-tern high concentrate feeding, the local RAS in mammary is activated, ratio of ACE/ACE 2 varies and the ACE 2-Ang1-7-MasR pathway takes the dominat position, that is to say that the anti-injury effect of ACE 2 becomes significant. ACE 2 helps to protect the mammary from inflammatory injuries by degrading Ang II to Ang1-7.

中图分类号:

S823.9⁺1.2

刘小倩，刘颖，荣超，王鲲，肖航，张源淑. ACE 2在奶牛乳腺中的表达定位及其抗炎性损伤作用的研究[J]. 畜牧兽医学报, 2017, 48(3): 552-560.

LIU Xiao-qian，LIU Ying, RONG Chao，WANG Kun，XIAO Hang，ZHANG Yuan-shu. Expression Localization of Angiotensin Converting Enzyme 2 (ACE 2) in Dairy Cow Mammary Gland and Its Anti-inflammation Effect[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(3): 552-560.

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