中文摘要：

试验预探索脂多糖（LPS）刺激奶牛乳腺上皮细胞炎性因子产生时间、浓度以及其对NF-κB信号通路的激活情况。用酶消化法培养奶牛乳腺上皮细胞（b MEC）;CCK-8法筛选LPS最佳刺激浓度（IR≤10%）,并用其处理对数期生长的b MEC。ELISA试剂盒检测0-24 h不同处理时间细胞培养液上清中TNF-α和IL-1β表达变化,以确定炎症模型是否建立成功;用Western-Blot检测核转录因子NF-κB（p-p65和p65）及其抑制蛋白（p-IκBα和IκBα）的表达。结果显示,用酶消化法成功培养原代b MEC;CCK-8法筛选LPS的最佳致炎浓度为1μg/m L;LPS处理后3-24 h可极显著增加奶牛乳腺上皮细胞中TNF-α和IL-1β表达,并在处理后15 min极显著提高b MEC中p-p65和p-IκBα的表达。上述结果说明,LPS能够在3-24 h内有效诱导b MEC炎症模型的建立,并能在15 min时活化奶牛乳腺上皮细胞中的NF-κB信号通路。

英文摘要：

The aims of this study were to explore the expression of inflammatory cytokines and activation of NF- κB signal pathway in primary bovine mammary epithelial cells（b MEC）after lipopolysaccharide（LPS）stimulation.The b MECs were enzymatically digested and method of CCK-8 was used to ascertain the suitable dose of LPS（IR≤10% as the standard）for stimulating the b MECs in their exponential growth phase.The concentrations of TNF-α and IL-1β in cellular supernatants were detected by ELISA kits at 0-24 h after LPS treatment to determinate the establishment of the cellular inflammation model.The expressions of NF-κB（p-p65 and p65）and（p-IκBα and IκBα）were detected by using Western-Blot.Our results showed that b MECs were cultured successfully by using enzymatic digestion method. The optimal concentration of LPS for inducing inflammation screened by using CCK-8 was 1 μg/m L.LPS significantly increased the expressions of both TNF-α and IL-1β at 3-24 h and p-p65 and p-IκBαat 15 min after treatment in b MECs.Our data suggest that LPS induces inflammation in b MECs at 3-24 h while the NF-κB signaling pathway is activated at 15 min after treatment.