LPS致奶牛蹄真皮细胞炎症模型的建立

Establishment of LPS-induced inflammation model in hoof dermal cells of dairy cows

为建立LPS致奶牛蹄真皮细胞炎症模型,通过分离、培养奶牛蹄真皮细胞,用不同浓度的LPS处理蹄真皮细胞,倒置显微镜下观察细胞形态,MTT法检测细胞活力,RT-PCR法检测CYP3A4、CYP1A1mRNA的表达,Western blot法检测CYP450蛋白的表达,并通过ELISA法检测细胞上清液中TNF-ɑ、IL-1β的含量,来确定LPS是否对细胞造成损伤。结果表明,随着LPS浓度的升高,对蹄真皮细胞的损伤也随之升高,CYP450蛋白的表达呈升高的趋势,在10μg/mL时表达最明显,10μg/mL LPS处理48h,对CYP3A4、CYP1A1mRNA表达的抑制作用较明显,模型组TNF-ɑ和IL-1β的含量极显著高于对照组(P<0.01)。本试验表明,10μg/mL LPS作用48h可建立可靠的蹄真皮细胞炎症模型。

To establish the LPS-induced inflammation model in hoof dermal cell,the primarily cultured dermal cells in hoof were exposed to different concentrations of LPS.The morphology of dermal cells was detected under inverted microscope.The dermal cells viability was determined by MTT assay.The expressions of CYP3A4,CYP1A1 and CYP450 were determined by RT-PCR and Western blot methods,respectively.The contents of TNF-ɑ,IL-1β in the supernatant of dermal cells were measured by the enzyme-linked immunosorbent assay（ELISA）method.It showed that,with the increase of LPS concentrations,the inhibition rate of cultured dermal cells increased,the expression of CYP450 protein increased and was most obvious at 10μg/mL.The expression of CYP3A4,CYP1A1 was most obvious with the treatment of 10μg/mL LPS for 48 h.The contents of TNF-ɑ,IL-1β in the model group was significantly higher than those in the control group（P0.01）.It shows that the inflammation model of dermal cells was established successfully with the treatment of 10 μg/mL LPS for 48 h.