本文關(guān)鍵詞：柑橘屬植物葉抗奶牛乳腺炎的機制研究　出處：《成都醫(yī)學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 奶牛乳腺炎 柑橘屬植物葉 抗炎 抑菌 抗氧化

【摘要】：目的:通過研究柑橘屬植物葉對奶牛乳腺上皮細(xì)胞炎癥模型的干預(yù),對金葡菌的體外抑制作用,對自由基的體外清除能力,探討其抗奶牛乳腺炎的作用機制。方法:(1)采用MTT比色法考察不同品種柑橘屬植物葉對奶牛乳腺上皮細(xì)胞(Bovine epithelial mammary cells,BMECs)的毒性作用及對細(xì)胞發(fā)生炎癥后的細(xì)胞活力的影響。利用金葡菌致炎蛋白構(gòu)建炎性奶牛乳腺上皮細(xì)胞模型,提取各實驗組BMECs總RNA,測定TNF-α、IL-1β、IL-6、SOD、ACACA、FASN、CSN2、CSN3等8種細(xì)胞因子的基因表達情況;測定在柑橘屬植物葉干預(yù)下,TNF-α、IL-1β、IL-6、NO等4種炎癥細(xì)胞因子的表達水平。(2)采用微量肉湯稀釋法測定不同品種柑橘屬植物葉對金葡菌(Staphylococcs aureus,S.aureus ATCC 29213)的最低抑菌濃度,考察各樣品的體外抑菌能力。(3)通過測定對自由基DPPH·和O2ˉ·的清除能力,考察不同品種柑橘屬植物葉的體外抗氧化能力;構(gòu)建柑橘屬植物葉的共有HPLC色譜條件,獲取各樣品的HPLC化學(xué)表達譜,進行聚類分析,并結(jié)合體外抗氧化數(shù)據(jù),采用逐步回歸法解析“譜-效”關(guān)系。結(jié)果:(1)不同品種柑橘屬植物葉對炎癥細(xì)胞模型的干預(yù)作用有明顯差異。紅橘葉的抗炎作用最強,能抑制主要炎癥因子TNF-α、IL-6、IL-1β基因的表達;顯著提高SOD基因的表達水平,其它樣品的抗炎作用未表現(xiàn)一致性,且部分樣品對炎性基因的影響不具有統(tǒng)計學(xué)意義(P0.05);紅橘葉不能增加乳汁成分ACACA、FASN、CSN2、CSN3基因的表達,部分雜交品種及柚類能顯著促進CSN2、CSN3基因的表達(P0.05)。(2)柑橘屬植物葉對S.aureus ATCC 29213的抑菌能力有較大差異,紅橘葉抑菌效果明顯,在濃度為29.6813mg/ml時能完全抑制細(xì)菌的生長。其余樣品的體外抑菌能力較弱。(3)體外抗氧化實驗中,柑橘屬植物葉對DPPH·的清除能力及對O2ˉ·的清除能力分別為2.08%~29.06%、0.4%~14.45%。HPLC圖譜顯示樣品的化學(xué)表達譜信息豐富,同一大類樣品有較高的相似度,不同大類之間區(qū)別較明顯;分別篩選出了對清除DPPH·和O2ˉ·貢獻最大的6個化學(xué)成分。結(jié)論:柑橘屬植物葉在抗炎、抗菌及抗氧化方面均未顯示較明顯效力,說明該三種機制并非是其防治奶牛乳腺炎的主要原因。所有樣品中,紅橘葉的抗炎及抗菌作用略優(yōu)于其他品種,顯示出傳統(tǒng)柑橘品種的優(yōu)勢,也提示選擇藥源時應(yīng)充分考慮品種因素。
[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- 偽, IL-1 尾, IL-6, SOD, ACACACAFASN, CSN2and CSN3, was determined. TNF- 偽 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- 偽 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.
【學(xué)位授予單位】：成都醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.23

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