摘要
本试验旨在研究催乳素(PRL)对奶牛乳腺上皮细胞(BMECs)中酪氨酸激酶2/信号传导及转录激活蛋白5(JAK2/STAT5)信号通路介导的乳铁蛋白(LF)合成和分泌的影响。添加不同浓度的PRL[0(对照)、10、100、1000 ng/mL]处理BMECs 24 h,分析PRL对LF含量和LF、催乳素受体(PRLR)以及JAK2/STAT5信号通路基因表达量和相关蛋白表达的影响。为了验证STAT5在PRL促进BMECs LF合成和分泌的关键作用,添加STAT5抑制剂匹莫齐特(Pimozide)[未添加Pimozide(对照)、10μmol/L Pimozide、100 ng/mL PRL、10μmol/L Pimo⁃zide+100 ng/mL PRL]处理BMECs 24 h,测定PRL和Pimozide对LF含量和LF、JAK2/STAT5信号通路基因表达量和相关蛋白表达以及相对荧光强度的影响。结果表明:1)与对照组相比,10、100、1000 ng/mL PRL能够显著或极显著提高BMECs上清液中的LF含量(P<0.05或P<0.01);Pimozide组能够极显著降低BMECs上清液中的LF含量(P<0.01);PRL组能够极显著提高BMECs上清液中的LF含量(P<0.01)。2)与对照组相比,10 ng/mL PRL组LF、PRLR、JAK2及STAT5基因相对表达量显著升高(P<0.05);100 ng/mL PRL组LF、PRLR、JAK2和STAT5基因相对表达量极显著升高(P<0.01);1000 ng/mL PRL组PRLR和STAT5基因相对表达量显著或极显著升高(P<0.05或P<0.01);Pimozide组LF基因相对表达量显著降低(P<0.05);PRL组BMECs中LF基因相对表达量显著升高(P<0.05)。3)100、1000 ng/mL PRL组BMECs中LF蛋白表达量极显著升高(P<0.01);10、100、1000 ng/mL PRL显著或极显著提高BMECs中p⁃JAK2/JAK2和p⁃STAT5/STAT5表达量(P<0.05或P<0.01);Pimozide组和Pimo⁃zide+PRL组极显著降低BMECs中LF蛋白表达量(P<0.01);PRL组极显著升高BMECs中LF蛋白表达量(P<0.01)。4)Pimozide组和Pimozide+PRL组显著或极显著降低BMECs中LF或p⁃STAT5的相对荧光强度(P<0.05或P<0.01);PRL组极显著升高BMECs中LF和p⁃STAT5的相对荧光强度(P<0.01)。综上所述,PRL通过JAK2/STAT5信号通路促进BMECs中LF合成和分泌。
The aim of this experiment was to investigate the effects of prolactin(PRL)on the synthesis and secretion of lactoferrin(LF)mediated by the Janus kinase 2/signal transducer and activator of transcription 5(JAK2/STAT5)signaling pathway in bovine mammary epithelial cells(BMECs).BMECs were treated with different concentrations of PRL[0(control),10,100,1000 ng/mL]for 24 h.The effects of PRL on LF content and the expression of LF,prolactin receptor(PRLR)and JAK2/STAT5 signaling pathway genes and related proteins were analyzed to verify the key role of STAT5 in prolactininduced LF synthesis and secretion in BMECs.BMECs were treated with STAT5 inhibitor Pimozide[no Pimozide(control),10μmol/L Pimozide,100 ng/mL PRL,and 10μmol/L Pimozide+100 ng/mL PRL]24 h,effects of PRL and Pimozide on LF content and gene and related protein expression and relative fluorescence intensity of LF and JAK2/STAT5 signaling pathway were determined.The results showed as follows:1)compared with the control group,10,100,1000 ng/mL PRL could significantly or very significantly increase the LF content in the cell supernatant of BMECs(P<0.05 or P<0.01);the Pimozide was able to significantly reduce the LF content in the cell supernatant of BMECs(P<0.01);the PRL was able to significantly increase the LF content in the supernatant of BMECs cells(P<0.01).2)The relative expression levels of LF,PRLR,JAK2 and STAT5 genes in the 10 ng/mL PRL group were significantly increased(P<0.05);the relative expression levels of LF,PRLR,JAK2 and STAT5 gene were very significantly higher in the 100 ng/mL PRL group(P<0.01);the relative expression levels of PRLR and STAT5 genes in the 1000 ng/mL PRL group were significantly or extremely significantly increased(P<0.05 or P<0.01);the relative expression level of LF gene in the Pimozide group decreased significantly(P<0.05);the relative expression level of LF gene in BMECs in the PRL group was significantly increased(P<0.05).3)The expression of LF protein in BMECs in the 100 and 1000 ng/mL PRL groups gradually increased(P<0.01);10,100,1000 ng/mL PRL significantly or very significantly increased the expression of pJAK2/JAK2 and pSTAT5/STAT5 in BMECs(P<0.05 or P<0.01);the Pimozide and the Pimozide+PRL significantly reduced the expression of LF protein in BMECs(P<0.01).LF protein expression was significantly increased in BMEC of PRL group(P<0.01).4)The Pimozide and Pimozide+PRL significantly or very significantly reduced the relative fluorescence intensity of LF or pSTAT5 in BMECs(P<0.05 or P<0.01);the relative fluorescence intensity of LF and pSTAT5 in BMECs was very significantly increased in the PRL group(P<0.01).In summary,PRL promotes PRL synthesis and secretion in BMECs cells of dairy cows through the JAK2/STAT5 signaling pathway.
作者
杨跃杰
连帅
武瑞
王建发
YANG Yuejie;LIAN Shuai;WU Rui;WANG Jianfa(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,China;Heilongjiang Provincial Key Laboratory of Prevention and Control of Bovine Diseases,Daqing 163319,China)
出处
《动物营养学报》
CAS
CSCD
北大核心
2024年第3期1916-1926,共11页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
国家自然科学基金项目(32172814,32172937,31772698)
黑龙江省自然科学基金项目(TD2022C005)。
关键词
乳铁蛋白
催乳素
信号通路
基因表达调控
lactoferrin
prolactin
signaling pathway
gene expression regulation
