| This study focused on the expression,localization and anti-injury effects of ACE2 in bovine mammary tissue.The research consists of two parts:1 Gene expression and cellular localization of ACE2 and ACE in bovine mammary glandThis study tried to figure out the expression and cellular localization of RAS in cow mammary tissue by RT-PCR?Western-Blot and immunofluorescence techniques.In this study,selected healthy lactating Holstein living mammary gland tissues and bovine mammary epithelial cells(Mac-T cells)performed the following tests:1)Using RT-PCR to determine the mRNA expression of ACE/ACE2 in cow mammary tissue;2)Determining the ACE2 gene segment by cloning and amplification;3)Determining the ACE/ACE2 protein expression in mammary tissue using Western-Blot method;4)Using immunofluorescence method to determine the cellular localization of ACE/ACE2 protein in bovine mammary epithelial cells.Results:1)RT-PCR results showed that both ACE and ACE2 gene were expressed in cow mammary tissue;2)ACE2 gene was cloned and identified,sequenced size of 202bp,in line with expectations;3)Western Blot results found specific bands of the ACE and ACE2 proteins,and confirmed the expression of two proteins;4)Immunofluorescence results proved that both ACE and ACE2 protein exeisted in the bovine mammary epithelial cells and mainly localized in the cytoplasm and membrane.This is the first time to confirm the presence of ACE2 and ACE in bovine mammary gland.This will provide evidence to further study the anti-injury effects of ACE2.2 The Anti-injury Effects of ACE2 on Mammary Tissue of Lactating Bovine Fed High Concentrate DietIn this study we selected 6 healthy lactating Holstein cows,and randomly divided them into normal control group(forage ratio 4:6)and high-concentrate group(forage ratio 6:4),single column fed regular meals,squeeze milk and milk samples were collected.After 20 weeks,we collected the living breast tissue from all experimental cows.the following tests were performed:1)Determining the number of milk somatic cells(SCC)?LPS content and the enzyme activities of a few enzymes including NAGase,MPO and AKP from milk and mammary tissures;2)Morphological examination of breast tissue;3)The mRNA expression of ACE,AT1,ACE2 and Mas in mammary tissues by the Real-Time PCR;4)Determining the contents of Ang ? and Angl-7 in mammary tissues by ELISA assay.Results:1)Compared with the control group,the milk somatic cell counts(SCC)?LPS content,the activities of NAGase and AKP in the milk from high-concentrate group all increased significantly(P<0.05);MPO had no significant differences between the two groups(P>0.05);2)HE staining showed that the high concentrate group of cow mammary tissue injury and inflammatory changes;3)Compared with the control group,the mRNA expression levels of the ACE,AT1,ACE2,Mas in high concentrate group also significantly increased(P<0.05),the ratio of ACE2/ACE mRNA expression in high concentrate group was more than that of the control group;3)Compared with normal control group,the content of Angl-7 in high-concentrate group was significantly increased(P<0.05);the content of Ang? significantly decreased(P<0.05).Conclusion:The mammary damages of lactating cows fed high-concentrate diet in a long-term may induced.The local RAS in mammary can be activated,and the activities of ACE2-Angl-7-Mas pathway is higher than the other pathway,Or the anti-injury effects of ACE2 is dominated,the ACE2 has an anti-injury effect or protective effect by degrading Ang ? to Angl-7... |
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