miR-15a/16a簇在金葡菌诱导的奶牛乳腺炎症反应中的调控作用及其分子机制

Mastitis is a common disease caused by a variety of pathogens. We have compared the miRNA expression profiling of the mammary tissues between healthy cow and Staphylococcus aureus (Staph. aureus) infected mastitis cow using miRNA chips and Q-PCR methods. The results showed that the expression of miR-15a/16a cluster was significantly downregulated in the mastitis tissues. We predicted the target gene for miR-15a/16a cluster by bioinformatics software, the result showed that the potential target gene are TNFSF13B, CXCL10 and CD163, which involved in the inflammatory response. Meanwhile it has been reported that miR-15a/16a participated in the regulation immune response by suppressing production of inflammatory mediators in the infected cells. Therefore we speculate that miR-15a/16a cluster may involve in mastitis inflammation response via regulating their expression of downstream targeted proteins and related inflammatory cytokines. To test the above hypothesis, several following experiments are needed to be performed: (1) to investigate the miR-15a/16a cluster histological localization and expression in the mammary tissues and neutrophils between healthy and Staph. aureus infected mastitis cows using the Q-PCR and in situ hybridization; (2) to detect the miRNA regulation function in the inflammation process of mammary epithelial cells which will be heat inactivated Staph. aureus and induced cells inflammation, on this basis, performing the over-expression, inhibiting expression, immunofluorescence and flow cytometry experiments; (3) to analyze the expression pattern of miR-15a/16a cluster members and compare the impact of miR-15a/16a cluster and single miR-15a, miR-16a on their downstream protein and inflammatory cytokine expressions, respectively. The regulatory mechanism of the miR-15a/16a cluster in mastitis inflammation response may be revealed, which would provide theoretical basis for mastitis treatment, resistance breeding in dairy cattle.

奶牛乳腺炎是由多种病原体感染引起的重要疾病。我们通过健康和金葡菌感染的乳腺炎奶牛乳腺组织miRNA表达芯片实验，发现并验证了乳腺炎奶牛的miR-15a/16a簇表达明显下调。我们推测miR-15a/16a簇可能通过调控下游TNFSF13B、CXCL10和CD163等免疫相关靶基因和炎症因子的表达，在乳腺炎症反应中发挥功能。拟通过Q-PCR和原位杂交定量和定位该miRNA簇在健康和金葡菌感染奶牛乳腺组织和中性粒细胞中的表达；利用热灭活的金葡菌诱导乳腺上皮细胞建立实验模型，在此基础上，采用基因过表达、抑制表达、免疫荧光、流式细胞等技术，检测该miRNA簇在炎症反应中的调控作用；分析miR-15a/16a簇成员的表达模式，比较该miRNA簇与单个miRNA对下游蛋白及炎症因子表达的影响。研究结果以期阐明miR-15a/16a簇在金葡菌诱导乳腺炎症反应中的调控机制，为奶牛乳腺炎抗病育种提供依据。

奶牛乳腺炎是由多种病原体感染引起的重要疾病。通过荧光定量和原位杂交检测技术，证实了bta-miR-15a/16a在健康奶牛和金葡菌感染的乳腺炎奶牛乳腺组织和血液中性粒细胞中存在表达差异。定量表达结果显示bta-miR-15a在乳腺炎牛乳腺组织和中性粒细胞中的表达量分别是健康牛乳腺组织中表达量的3.69倍和6.84倍（P<0.05），bta-miR-16a在乳腺炎牛乳腺组织中的表达量是健康牛乳腺组织中表达量的2.47倍和13.75倍（P<0.05）。原位杂交结果表明bta-miR-15a和bta-miR-16a在奶牛乳腺腺泡包膜均有表达，而且乳腺炎牛乳腺组织中的表达量较多。利用生物信息学软件和载体构建、双荧光素酶测定等实验验证，筛选出bta-miR-15a/16a的关键靶基因CD163，bta-miR-15a、bta-miR-16a可显著降低CD163基因的荧光素酶活性，并且bta-miR-15a/16a簇相对单个bta-miR-15a和bta-miR-16a在调控下游靶基因CD163表达方面作用效力更强。研究结果初步阐明了bta-miR-15a/16a簇在金葡菌诱导乳腺炎症反应中的调控机制，为奶牛乳腺炎抗病育种提供了理论依据。