奶牛LBP基因功能位点变异调节乳腺上皮细胞对GN菌易感性的分子机制

Gram-negative （GN）bacteria are responsible for approximately 30%-50% clinical cases of bovine mastitis, which leads to immeasurable economic loss to the farmers. Lipopolysaccharide(LPS) is one the the most important pathogenic components in the cell membrance. Lipopolysaccharide-binding protein(LBP) plays a vital role in the innate immune recognition of GN bacterium. In the inflammatory signal transmission, the process LBP recognizes and binds with LPS via LPS-binding sites is the first prelude, but little is known about LBP mutations and their effects on cows' susceptibility to mastitis. The dual functions of LBP (inflammation and anti- inflammation) are dependent on the LBP concentration in the blood, specific structure and functional sites. The clarification of the polymorphism in the LBP functional sites is the precondition of understanding the GN bacterium-induced mastitis. We have found SNPs in the functional sites of LBP gene significantly change the susceptibility to mastitis in dairy cows for the first time. It can be divided into LBP1-5. To investigate the real significance of these mutations in vivo and in vitro, LBP1-5 molecules are used to conduct the following studies. LBP1-5 molecules with different concentration were added into the medium of LPS-induced bovine mammary epithelial cells; inflammation signal molecules are analyzed to screen and decide the optimal LBP molecule.The blood and milk samples of different genotypic individuals are collected to explore the association between the mutations and expression levels of inflammation signal molecules. The animal mastitis models induced by LPS injection in mammary gland are also used to study the functions of different LBP molecules. Taken together, it will be beneficial for the clinical researches and feasible for us to breed mastitis-resistant cows in the future.

约30%-50%临床奶牛乳腺炎是革兰氏阴性菌引起的，LPS是细菌胞膜高度保守致病组分，识别LPS是介导炎症和抗炎反应的首要环节。LBP对LPS 具有高亲和性，可识别和结合LPS，在固有免疫中发挥至关重要的作用。LBP发挥致炎和抗炎双重作用有赖于血液中LBP浓度、独特的分子结构及功能位点，查明LBP功能位点变异是了解GN菌诱发奶牛乳腺炎机制的前提。本研究组首次从牛群中检出LBP重要功能区变异，有5种LBP变异类型，显著改变乳腺炎发病率。为了揭示其调控机制，本研究通过不同浓度和分子结构LBP处理乳腺上皮细胞，检测LPS致炎信号分子表达；采集不同LBP基因型乳腺炎奶牛的外周血及奶样，测定LBP和sCD14相关致炎信号分子浓度；体内、外筛选和验证致炎作用较弱的LBP变异类型；通过LPS诱导乳腺炎奶牛模型，评价LBP变异对乳腺炎的作用。该研究为利用LBP进行临床研究及乳腺炎抗病育种提供理论依据。

约30%-50%临床奶牛乳腺炎是革兰氏阴性菌引起的，LPS是细菌胞膜高度保守致病组分，识别LPS是介导炎症和抗炎反应的首要环节。LBP对LPS 具有高亲和性，可识别和结合LPS，在固有免疫中发挥至关重要的作用。另外LBP发挥致炎和抗炎双重作用有赖于血液中LBP浓度、独特的分子结构及功能位点。本研究首次从牛群中检出LBP重要功能区的4种LBP变异类型。为了揭示不同变异类型的调控机制，本研究通过利用不同浓度和分子结构LBP处理乳腺上皮细胞，检测LPS致炎信号分子表达；采集不同LBP基因型乳腺炎奶牛的外周血及奶样，测定LBP和sCD14相关致炎信号分子浓度；体内、体外筛选和验证致炎作用较强的LBP变异类型；通过LPS诱导乳腺炎奶牛模型，评价LBP变异对乳腺炎的作用。通过本研究，得到如下重要结果：（1）在体外乳腺上皮细胞炎症模型中，LBP参与LPS诱导乳腺上皮细胞炎症反应的TLR4/NF-KB信号通路；（2）临床乳腺炎活体奶牛中，第三外显子g.4619G → A变异的奶牛，其血样和乳样中炎症因子的浓度显著高于野生型或LBP其他位点变异的奶牛；（3）野生型和变异的LBP蛋白在低浓度时能够增敏LPS诱导的奶牛乳腺炎症反应，而高浓度的LBP变异蛋白可以有效缓解LPS诱导的奶牛乳腺炎；（4）与野生型蛋白比较，在3个变异蛋白中，外显子3变异的LBP蛋白MU-LBP2（g.4619G → A）在低浓度时致炎作用最强，在高浓度时抑炎作用最强。本研究的主要科学意义在于：（1）为奶牛乳腺炎抗病育种提供了理论参照。通过敲减或增强第三外显子突变（g.4619G → A）奶牛体内LBP蛋白的表达，有望达到双重降低乳腺炎发病的效果。即敲减第三外显子突变健康奶牛LBP蛋白表达，减少奶牛群体对致病菌的敏感性；增强第三外显子突变患乳腺炎奶牛LBP蛋白表达，使高剂量的LBP发挥抑制致病菌的作用。（2）为开发抑制奶牛乳腺炎的靶向药物提供实验依据。本研究发现由大肠杆菌诱导的奶牛乳腺炎是通过激活TLR4/NF-κB信号通路而发生的，开发抑制该通路关键点激活的靶向药物，对从源头上抑制乳腺炎的发生有潜在的应用价值。