【摘要】：胸膜肺炎放線桿菌是無芽孢多形性球狀短桿菌,能引起豬呼吸系統(tǒng)疾病,具有高死亡和高傳染等特點,臨床上主要表現(xiàn)為肺部炎癥和呼吸系統(tǒng)功能障礙,常呈暴發(fā)性流行,并常與多種細(xì)菌混合感染。鹽酸小檗堿是從黃連等植物中提取的季胺型異喹啉類生物堿,對多種革蘭氏陽性、陰性菌均有較好的抑制和殺滅效果。本文擬研究鹽酸小檗堿對該菌的抑制和殺滅效果并初步闡明作用機制。主要研究結(jié)果如下：1.鹽酸小檗堿的體外抑菌效果的研究：采用二倍稀釋法,測定鹽酸小檗堿對豬胸膜肺炎放線桿菌的最低抑菌濃度(Minimal inhibitory concentration,MIC)和最小殺菌濃度(Minimal bactericidal concentration,MBC),并繪制其抑菌曲線。結(jié)果表明：鹽酸小檗堿對豬胸膜肺炎放線桿菌具有較好的體外殺滅效果,其MIC和MBC均為0.3125 mg/mL；當(dāng)鹽酸小檗堿濃度為MIC時,細(xì)菌在8 h內(nèi)全部死亡,濃度為1/2 MIC及以下時,細(xì)菌生長受到明顯抑制,但生長曲線與正常組相似,表明鹽酸小檗堿對豬胸膜肺炎放線桿菌存在劑量依賴而非時間依性。以上結(jié)果表明鹽酸小檗堿對豬的胸膜肺炎放線桿菌具有較好的體外殺菌效果。2.鹽酸小檗堿對豬胸膜肺炎放線桿菌細(xì)胞膜的影響：通過測定大分子物質(zhì)和Ca,Mg,K離子的泄漏判斷細(xì)胞膜的完整性,并對膜上蛋白進(jìn)行熒光分析；測定還原糖的利用情況和采用氮苯基-1-萘胺(N-Phenyl-1-Napthylamine,NPN)法判斷細(xì)胞膜的滲透性；通過測量乳酸脫氫酶(Alkaline phosphatase,LDH)和堿性磷酸酶(Alkaline phosphatase,AKP)的體外活性判斷藥物對細(xì)菌細(xì)胞壁完整性的影響。結(jié)果表明：鹽酸小檗堿作用于細(xì)菌后,在前10 min內(nèi),大分子物質(zhì)DNA胞外濃度變化不顯著,1-12h內(nèi),胞外DNA濃度和蛋白質(zhì)濃度先上升后下降,Ca,Mg,K離子胞外濃度升高,離子泄露,表明鹽酸小檗堿能夠破壞細(xì)胞膜的完整性和滲透性。胞外LDH和AKP的活性與對照組相比變化不明顯。膜蛋白熒光結(jié)果顯示,鹽酸小檗堿能與細(xì)胞膜上的色氨酸殘基相結(jié)合,引起細(xì)胞膜結(jié)構(gòu)發(fā)生改變,其淬滅方式為靜態(tài)淬滅。以上結(jié)果表明鹽酸小檗堿能夠影響胸膜肺炎放線桿菌細(xì)胞膜的完整性和滲透性,與膜上的某些蛋白質(zhì)結(jié)合從而影響蛋白質(zhì)的結(jié)構(gòu)與功能。3.鹽酸小檗堿對豬胸膜肺炎放線桿菌蛋白質(zhì)表達(dá)的影響：通過測定細(xì)菌總蛋白含量的變化及SDS-聚丙烯酰胺凝膠電泳(Sodium dodecyl sμLphate polyacrylamide gel-electrophoresis,SDS-PAGE)測定細(xì)菌蛋白表達(dá)的差異,研究鹽酸小檗堿對細(xì)菌蛋白質(zhì)合成的影響。結(jié)果表明：鹽酸小檗堿引起細(xì)菌總蛋白的表達(dá)量先升高后降低正常組蛋白質(zhì)條帶多而清晰,藥物作用后蛋白表達(dá)出現(xiàn)差異,一些蛋白條帶變暗甚至消失,推測44.3kDa以下的蛋白質(zhì)可能為鹽酸小檗堿的作用靶點。以上結(jié)果表明鹽酸小檗堿使得細(xì)菌蛋白質(zhì)的合成和表達(dá)下降。4.鹽酸小檗堿對豬胸膜肺炎放線桿菌DNA合成的影響：通過4',6-二脒基-2-苯基吲哚(4'、6-diamidino-2-phenylindole,DAPI)染色,紫外吸收光譜,瓊脂糖凝膠電泳,EB-DNA(溴化乙錠-DNA)體系,熱溶解曲線等方法檢測細(xì)菌DNA在藥物作用前后表達(dá)量的變化及DNA與鹽酸小檗堿的體外結(jié)合狀態(tài),研究鹽酸小檗堿對細(xì)菌DNA的影響。熒光染色結(jié)果表明,正常組熒光強度較強,數(shù)目較多，藥物作用組熒光強度降低,表明鹽酸小檗堿能夠影響細(xì)菌DNA的表達(dá)。紫外吸收光譜的改變和瓊脂糖凝膠電泳結(jié)果表明,鹽酸小檗堿能夠與DNA結(jié)合。EB-DNA和熱溶解曲線結(jié)果表明鹽酸小檗堿能夠與DNA在體外結(jié)合,其結(jié)合方式與EB相同但作用強度更高。以上結(jié)果表明鹽酸小檗堿能夠影響細(xì)菌DNA的表達(dá),在體外與DNA結(jié)合,其結(jié)合方式為嵌插作用。綜合以上結(jié)果,鹽酸小檗堿對豬的胸膜肺炎放線桿菌具有良好的殺滅效果,作用機理為引起細(xì)菌細(xì)胞膜完整性的改變,影響細(xì)菌蛋白質(zhì)和DNA的合成。
[Abstract]:Actinobacillus pleuropneumoniae is a non spore forming * short form Bacillus brevis, which can cause respiratory diseases in pigs. It is characterized by high mortality and high infection. Clinically, it is mainly manifested as pulmonary inflammation and respiratory dysfunction. It is often shown as an outbreak epidemic and is often mixed with a variety of bacteria. Berberine hydrochloride is extracted from plants such as Coptis chinensis. Quaternary isoquinoline alkaloids have good inhibitory and killing effects on a variety of gram-positive and gram-negative bacteria. In this paper, the inhibitory and killing effects of berberine hydrochloride on the bacteria were studied and its mechanism was preliminarily elucidated. The main results are as follows: 1. In vitro inhibitory effects of berberine hydrochloride on bacteria were studied by double dilution method. Berberine hydrochloride * Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (Minimal bactericidal concentration, MBC) of Actinobacillus pleuropneumoniae, and their bacteriostatic curves were drawn. The results showed that berberine hydrochloride had a good in vitro killing effect on * swine Actinobacillus pleuropneumoniae, MIC and MBC. They were all 0.3125 mg/mL; when the berberine hydrochloride concentration was MIC, the bacteria died within 8 h, and the growth of bacteria was significantly inhibited when the concentration was 1/2 MIC and below. However, the growth curve was similar to that of the normal group * indicating that berberine hydrochloride had a dose dependence rather than time dependence on the swine Actinobacillus pleuropneumoniae. * the effect of.2. berberine on the cell membrane of Actinobacillus pleuropneumoniae * cell membrane was determined by measuring the leakage of porcine Actinobacillus pleuropneumoniae. The integrity of the membrane was determined by measuring the leakage of macromolecules and Ca, Mg and K ions, and fluorescence analysis of the protein on the membrane was performed. The utilization of reducing sugar and nitrogen benzol were determined. The permeability of cell membrane was determined by N-Phenyl-1-Napthylamine (NPN) method, and the effect of the drug on the integrity of cell wall was evaluated by measuring the activities of lactate dehydrogenase (LDH) and alkaline phosphatase (AKP). Within 1-12 hours, the concentration of extracellular DNA and protein increased first and then decreased. The concentration of Ca, Mg, K ions increased and the ion leakage showed that berberine hydrochloride could destroy the integrity and permeability of cell membrane. The activity of extracellular LDH and AKP had no significant change compared with the control group. The results showed that berberine hydrochloride could bind to the tryptophan residues on the cell membrane, resulting in the change of cell membrane structure, and the quenching mode was static quenching. Effects of berberine hydrochloride and function.3. * on the protein expression of Actinobacillus pleuropneumoniae protein: the changes of bacterial total protein content and SDS- dodecyl Sodium Lphate polyacrylamide gel-electrophoresis (SDS-PAGE) were used to detect the difference of bacterial protein expression, and the effects of berberine hydrochloride on bacterial protein expression were studied. The results showed that the expression of total bacterial protein increased first and then decreased in normal group. The protein expression was different after the drug action. Some protein bands became dark or even disappeared. It was speculated that the protein below 44.3 kDa might be the target of berberine hydrochloride. The results showed that berberine hydrochloride reduced the protein synthesis and expression of.4. * the effect of berberine hydrochloride on the DNA synthesis of Actinobacillus pleuropneumoniae DNA: 4', 6- two amipryl -2- phenyl indole (4', 6-diamidino-2-phenylindole, DAPI) staining, UV absorption spectrum, agarose gel electrophoresis, EB-DNA (ethidium bromide -DNA) system, thermal dissolution. The effect of berberine hydrochloride on bacterial DNA was studied. The results of fluorescence staining showed that the fluorescence intensity of normal group was stronger, the number of bacterial DNA was larger, the fluorescence intensity of drug group was lower, indicating that berberine hydrochloride could affect bacterial DN. The results of ultraviolet absorption spectroscopy and agarose gel electrophoresis showed that berberine hydrochloride could bind to DNA. The results of EB-DNA and thermolysis curves showed that berberine hydrochloride could bind to DNA in vitro, and the binding mode was the same as that of EB, but the binding intensity was higher. In combination with DNA in vitro, the binding mode is inserted. In conclusion, berberine hydrochloride has good killing effect on * Actinobacillus pleuropneumoniae, and the mechanism of action is to cause changes in the integrity of bacterial cell membrane and affect the synthesis of bacterial protein and DNA.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S858.28

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