【作者】 徐佳佳；

【导师】 肖向红；

【作者基本信息】 东北林业大学 ， 生理学， 2010， 硕士

【摘要】 两栖类作为由水生过渡到陆生的物种,其生境中微生物区系十分复杂,在长期进化过程中两栖类皮肤逐步建立了通过皮肤分泌抗菌肽等物质以有效抵御环境中病原微生物侵袭的天然免疫防御体系。但近年来发现两栖类种群数量呈全球性急剧减少或消失趋势,认为导致这一现象的重要原因之一是两栖类感染性疾病。然而目前对于两栖类皮肤抗菌肽与环境微生物之间的关系尚不清楚。本研究以东北地区两栖类代表物种东北林蛙(Rana dybowskii)为试验对象,实验分6～72h、1～23d两个观察时段。6～72h时段,设4组：E.coli菌浴组、E.coli淋巴囊注射组、S.aureus菌浴组、S.aureus淋巴囊注射组。1～23d时段,采用温和电刺激法排空腺体内的分泌物后,设E.coli菌浴组和LB培养基组。通过对不同细菌胁迫下的不同时段东北林蛙皮肤分泌物的蛋白多肽浓度测定、RP-HPLC多肽图谱分析、抑菌活性和溶血活性分析等,观察其皮肤分泌物的特征和抗菌活性变化,以期揭示两栖类动物抵御环境微生物时皮肤分泌抗菌肽的动态变化规律。结果显示,胁迫前东北林蛙皮肤分泌物(正常对照组)RP-HPLC分离获得26个洗脱峰,显示出较强的广谱抗菌活性,尤以对G+细菌活性抑制更显著,低溶血活性。胁迫6～72h后,RP-HPLC多肽图谱显示,洗脱峰数量无明显变化,但峰C～K呈特异性变化,除峰C、D和E呈现波动变化外,峰F、G和H在胁迫后峰面积值均明显上升,峰I和K在胁迫后峰面积值均明显下降。东北林蛙皮肤分泌物蛋白浓度和抑菌活性均呈时间依赖性增强趋势。胁迫1-23d后,RP-HPLC多肽图谱显示,1～18d皮肤分泌物洗脱峰数量明显减少至4-10个,至23d基本恢复至胁迫前水平,其中A、B、C和D这4个洗脱峰持续出现,并具有较强的抑制G-和G+菌活性。蛋白多肽浓度第1天显著下降,1～7dE.coli菌浴组显著高于LB组,而13～18d则相反。东北林蛙分泌物排空后至少需要23天才可恢复到胁迫前水平,在此期间分泌物仍具有抗菌活性。本研究结果表明,东北林蛙蛙皮肤分泌物中抗菌肽作为皮肤免疫防御体系的重要组成,对环境中不同微生物胁迫刺激可提高其抗菌肽抗菌活性,并表现出非特异性应答反应,但对致病性较强的S.aureus更为敏感。该研究结果为进一步揭示东北林蛙皮肤分泌物的作用方式及变化规律提供了重要的理论和实验依据。更多还原

【Abstract】 Amphibians are the transitional species from the aquatic to the terrestrial. Their skin has gradually established a defensive natural immune defense system in the long-term evolution, to remove pathogenic microorganisms in the environment. But it has been found that amphibian populations were drastically reduced or disappeared global trend in recent years, and one of the important reasons of this phenomenon is infectious disease of amphibians. However, the relationship between environmental microorganisms and amphibian skin antimicrobial peptides is not clear.To reveal the changes of amphibian skin secretions under microbial challenges, the brown frog (Rana dybowskii) was selected as the test object, and the test subjects were divided into 6～72h,1～23d two observation periods.6～72h period, with 4 groups:E.coli bath group, E.coli sac injection group, S.aureus bacteria bath group and S.aureus sac injection group.1～23d period, after using a mild electrical stimulation emptying the secretion of skin gland, divided into E.coli bath group and LB medium group. Results showed by RP-HPLC analysis, protein concentration and antibacterial activity comparison. Observe the features and antimicrobial activity of skin secretions to reveal dynamics of secretion of amphibian skin against environmental microorganisms.The RP-HPLC results showed that normal skin secretions of R. dybowskii (control group) has isolated 26 peaks, indicating a strong broad-spectrum antimicrobial activity, particularly against G+ bacteria, and had low hemolytic activity.After challenged 6～72h, RP-HPLC peptide profiles showed no significant changes in the number of elution peaks, but peak C-K showed a specific change, in addition to peaks C, D and E showed fluctuation, the peak area of F, G and H significantly increased, peak I and K were significantly decreased. The antibacterial activity and protein concentration of skin secretions of R. dybowskii showed a time-dependent increasing trend.After challenged 1～23d, RP-HPLC peptide profiles displayed that 1～18d skin secretions significantly reduced and the number of elution peaks down to 4-10, restored to the level before challenged to 23d. And peaks A, B, C and D were persistent and had strong inhibition of G- and G+ bacteria. Peptide concentrations decreased significantly on day 1, and 1～7d E.coli bath groups were significantly higher than the LB bath groups, but 13-18d were opposite. Restoring the secretions of R. dybowskii to the level of unchallenged needed at least 23 days, as secretion still had antibacterial activity.This study indicated that antimicrobial peptides of skin secretions of R. dybowskii as the major components of skin immune defense system can induce non-specific immunity when facing different environmental stress, especially more sensitive to S.aureus. The results provide important theoretical and experimental basis for mode of skin secretions of R. dybowskii changes in further.更多还原