柑橘属植物叶抗奶牛乳腺炎的机制研究

发布时间：2018-01-13 13:26

本文关键词：柑橘属植物叶抗奶牛乳腺炎的机制研究　出处：《成都医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:通过研究柑橘属植物叶对奶牛乳腺上皮细胞炎症模型的干预,对金葡菌的体外抑制作用,对自由基的体外清除能力,探讨其抗奶牛乳腺炎的作用机制。方法:(1)采用MTT比色法考察不同品种柑橘属植物叶对奶牛乳腺上皮细胞(Bovine epithelial mammary cells,BMECs)的毒性作用及对细胞发生炎症后的细胞活力的影响。利用金葡菌致炎蛋白构建炎性奶牛乳腺上皮细胞模型,提取各实验组BMECs总RNA,测定TNF-α、IL-1β、IL-6、SOD、ACACA、FASN、CSN2、CSN3等8种细胞因子的基因表达情况;测定在柑橘属植物叶干预下,TNF-α、IL-1β、IL-6、NO等4种炎症细胞因子的表达水平。(2)采用微量肉汤稀释法测定不同品种柑橘属植物叶对金葡菌(Staphylococcs aureus,S.aureus ATCC 29213)的最低抑菌浓度,考察各样品的体外抑菌能力。(3)通过测定对自由基DPPH·和O2ˉ·的清除能力,考察不同品种柑橘属植物叶的体外抗氧化能力;构建柑橘属植物叶的共有HPLC色谱条件,获取各样品的HPLC化学表达谱,进行聚类分析,并结合体外抗氧化数据,采用逐步回归法解析“谱-效”关系。结果:(1)不同品种柑橘属植物叶对炎症细胞模型的干预作用有明显差异。红橘叶的抗炎作用最强,能抑制主要炎症因子TNF-α、IL-6、IL-1β基因的表达;显著提高SOD基因的表达水平,其它样品的抗炎作用未表现一致性,且部分样品对炎性基因的影响不具有统计学意义(P0.05);红橘叶不能增加乳汁成分ACACA、FASN、CSN2、CSN3基因的表达,部分杂交品种及柚类能显著促进CSN2、CSN3基因的表达(P0.05)。(2)柑橘属植物叶对S.aureus ATCC 29213的抑菌能力有较大差异,红橘叶抑菌效果明显,在浓度为29.6813mg/ml时能完全抑制细菌的生长。其余样品的体外抑菌能力较弱。(3)体外抗氧化实验中,柑橘属植物叶对DPPH·的清除能力及对O2ˉ·的清除能力分别为2.08%~29.06%、0.4%~14.45%。HPLC图谱显示样品的化学表达谱信息丰富,同一大类样品有较高的相似度,不同大类之间区别较明显;分别筛选出了对清除DPPH·和O2ˉ·贡献最大的6个化学成分。结论:柑橘属植物叶在抗炎、抗菌及抗氧化方面均未显示较明显效力,说明该三种机制并非是其防治奶牛乳腺炎的主要原因。所有样品中,红橘叶的抗炎及抗菌作用略优于其他品种,显示出传统柑橘品种的优势,也提示选择药源时应充分考虑品种因素。
[Abstract]:Objective: to study the inhibitory effect of citrus leaves on bovine mammary epithelial cell inflammatory model in vitro and the ability of scavenging free radicals in vitro. Methods MTT colorimetric assay was used to investigate the effects of different citrus leaves on dairy mammary epithelial cells (MEC). Bovine epithelial mammary cells. The toxic effect of BMECs and its effect on cell viability after inflammation were studied. The inflammatory dairy cow mammary epithelial cell model was constructed by using Staphylococcus aureus inflammatory protein and the total RNA of each experimental group was extracted. The gene expression of 8 cytokines, such as TNF- 伪, IL-1 尾, IL-6, SOD, ACACACAFASN, CSN2and CSN3, was determined. TNF- 伪 and IL-1 尾 IL-6 were measured under the intervention of citrus leaves. The expression level of four kinds of inflammatory cytokines, no, et al.) the trace broth dilution method was used to detect Staphylococcs aureus in leaves of different citrus plants against Staphylococcs aureus. The minimum inhibitory concentration of S.aureus ATCC 29213) was determined by the method of scavenging free radical DPPH 路and O _ 2 ~ (2 +). The in vitro antioxidant capacity of different citrus leaves was investigated. The common HPLC chromatographic conditions of citrus leaves were constructed, the HPLC chemical expression profiles of each sample were obtained, cluster analysis and in vitro antioxidant data were carried out. Stepwise regression method was used to analyze the "spectrum effect" relationship. Results there were significant differences in the effects of different citrus leaves on inflammatory cell model, but the anti-inflammatory effect of red orange leaf was the strongest. It can inhibit the expression of IL-1 尾 gene of TNF- 伪 and IL-6; The expression level of SOD gene was significantly increased. The anti-inflammatory effect of other samples was not consistent, and the effect of some samples on inflammatory gene was not statistically significant (P 0.05). The leaf of red orange could not increase the expression of CSN3 gene in the milk component ACACACAA FASN _ 2 and CSN _ 3, and some hybrids and grapefruit could significantly promote the expression of CSN2. The bacteriostatic ability of leaves of citrus plants to S.aureus ATCC 29213 was significantly different, and the bacteriostatic effect of red orange leaves was obvious. At the concentration of 29.6813 mg / ml, the growth of bacteria was completely inhibited. The antibacterial ability of the other samples was weak in vitro. The scavenging ability of citrus leaves to DPPH 路and to O _ 2 路were 2.08% and 29.06%, respectively. The HPLC chromatogram showed that the chemical expression profiles of the samples were rich in information, the samples of the same large class had high similarity, and the differences between the different groups were obvious. Six chemical constituents which contributed most to the scavenging of DPPH 路and O _ 2 路were screened out respectively. Conclusion: the leaves of Citrus have no obvious effect on anti-inflammatory, antibacterial and anti-oxidation. The results showed that these three mechanisms were not the main reason for the prevention and treatment of cow mastitis. In all samples, the anti-inflammatory and antibacterial effects of red orange leaves were slightly superior to those of other varieties, showing the advantages of traditional citrus varieties. It was also suggested that the variety factors should be fully considered when selecting the drug source.
【学位授予单位】：成都医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

【相似文献】