咖啡酸抗LPS诱导的犬子宫内膜上皮细胞炎性损伤作用

发布时间：2018-04-03 20:29

本文选题：咖啡酸　切入点：脂多糖　出处：《甘肃农业大学》2015年硕士论文

【摘要】：犬子宫内膜炎是宠物临床上常见的一种产科疾病,严重威胁宠物的健康。咖啡酸是一种具有抗菌抗炎作用的酚酸类化合物。本文通过咖啡酸抗LPS诱导的犬子宫内膜上皮细胞炎症试验,探讨咖啡酸的抗炎作用效果,为临床治疗犬子宫内膜炎的新药研发提供理论依据。本论文通过酶消法分离培养犬子宫内膜上皮细胞;通过MTT方法检测LPS对犬子宫内膜上皮细胞细胞活力的影响,从而筛选出诱导犬子宫内膜上皮细胞炎症模型的浓度和时间条件;通过MTT方法进行咖啡酸的药物毒性试验,确定咖啡酸的安全的剂量范围和时间,并选择出高、中、低三个咖啡酸剂量;通过MTT方法测不同剂量咖啡酸对LPS诱导的细胞活力的影响;通过ELISA方法检测不同剂量咖啡酸对LPS诱导的细胞培养液中炎性因子水平的影响;通过一氧化氮试剂盒检测细胞培养液中NO浓度的变化;通过AO和EB荧光染料双染细胞方法,检测咖啡酸对LPS诱导的细胞凋亡的影响。取得试验结果如下:LPS能够显著降低细胞活力,且呈时间剂量依赖性,50μg/m L LPS作用12 h成功诱导了细胞炎症模型;咖啡酸作用12 h后,1 ng/m L~200μg/m L组抑制率与对照组相比没有显著差异;咖啡酸作用24 h后,1 ng/m L~25μg/m L组活力无显著下降。在安全范围内选择25μg/m L、50μg/m L、100μg/m L作为咖啡酸低、中、高剂量。用中、高咖啡酸预处理3 h,细胞活力与对照组相比无显著差异,比模型组显著升高。模型组NO、IL-6、TNF-α水平与对照组相比显著升高;而咖啡酸组则出现不同程度的下降。AO/EB染色后,在荧光显微镜下能够观察到对照组多数为活细胞,模型组有大量凋亡细胞,而咖啡酸组凋亡细胞明显少于模型组。结果表明,咖啡酸具有一定的抗LPS诱导的犬子宫内膜上皮细胞炎性损伤作用。
[Abstract]:Endometritis is a common obstetrical disease in pets, which is a serious threat to the health of pets.Caffeic acid is a phenolic acid compound with antibacterial and anti-inflammatory effects.In this paper, the anti-inflammatory effect of caffeic acid on canine endometrial epithelial cells induced by LPS was studied, which provides a theoretical basis for the development of new drugs for the treatment of canine endometritis.In this paper, the canine endometrial epithelial cells were isolated and cultured by enzyme digestion, the effects of LPS on the viability of canine endometrial epithelial cells were detected by MTT method, and the concentration and time conditions of the inflammatory model of canine endometrial epithelial cells were screened out.The drug toxicity test of caffeic acid was carried out by MTT method. The safe dose range and time of caffeic acid were determined, and three doses of caffeic acid were selected as high, middle and low doses.The effects of different doses of caffeic acid on the cell viability induced by LPS were measured by MTT method, and the levels of inflammatory factors in cell culture medium induced by LPS were detected by ELISA method.The changes of no concentration in cell culture medium were detected by nitric oxide kit, and the effects of caffeic acid on apoptosis induced by LPS were detected by AO and EB fluorescent dye double staining cell methods.The results were as follows: lipopolysaccharide could significantly reduce cell viability, and the inflammatory model was induced in a dose-dependent manner by 50 渭 g / mL LPS for 12 h, but there was no significant difference in the inhibition rate of 1 ng/m L ~ (200 渭 g / mL) group compared with the control group after 12 h of caffeic acid treatment.There was no significant decrease in activity of 1 ng/m / L 25 渭 g / mL group treated with caffeic acid for 24 h.In the safe range, 25 渭 g / mL 50 渭 g / mL 50 渭 g / mL 100 渭 g / mL caffeic acid was selected as low, medium and high dose of caffeic acid.After treated with high caffeic acid for 3 h, the cell viability was not significantly different from that of the control group, and was significantly higher than that of the model group.Compared with the control group, the level of NO-IL-6 TNF- 伪 in the model group was significantly higher than that in the control group, while in the caffeic acid group, it was observed that most of the cells in the control group were living cells, and a large number of apoptotic cells were found in the model group.The apoptotic cells in caffeic acid group were significantly lower than those in model group.The results showed that caffeic acid could inhibit the inflammatory injury induced by LPS in canine endometrial epithelial cells.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.292

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