CAJ | 학술논문

本文旨在构建沙门菌-斑马鱼感染模型，研究幼年斑马鱼体内的异源自噬。用不同浓度鼠伤寒沙门菌浸染受精后72 h的幼年斑马鱼，绘制7 d内斑马鱼的存活率曲线以确定适宜的细菌感染量。以上述细菌量感染斑马鱼后，荧光显微镜下观察沙门菌在体内的播散情况；在不同时间点计数细菌菌落，并用蛋白免疫印迹法检测自噬相关蛋白 Lc3和 p62的表达，同时在电子显微镜下观察自噬体结构。结果显示，以1×109 CFU/ml细菌量感染斑马鱼后，2 d内斑马鱼存活率为100％，第3天开始出现死亡。荧光显微镜下发现，感染后4h鱼体中有细菌侵入，3d后细菌扩散至全身。细菌计数结果显示，整条鱼感染的细菌量随时间延长不断增加，但感染后10 h侵入鱼体细胞中的菌量显著低于8 h。蛋白免疫印迹结果显示，感染后8 h Lc3-Ⅱ表达显著升高，p62表达显著降低。透射电子显微镜下，感染后8 h幼鱼细胞中可观察到自噬体和自噬溶酶体结构。以上结果提示，用鼠伤寒沙门菌浸染幼年斑马鱼构建的模型可用于追踪病原菌感染引起的异源自噬的动态变化。
본문지재구건사문균-반마어감염모형，연구유년반마어체내적이원자서。용불동농도서상한사문균침염수정후72 h적유년반마어，회제7 d내반마어적존활솔곡선이학정괄의적세균감염량。이상술세균량감염반마어후，형광현미경하관찰사문균재체내적파산정황；재불동시간점계수세균균락，병용단백면역인적법검측자서상관단백 Lc3화 p62적표체，동시재전자현미경하관찰자서체결구。결과현시，이1×109 CFU/ml세균량감염반마어후，2 d내반마어존활솔위100％，제3천개시출현사망。형광현미경하발현，감염후4h어체중유세균침입，3d후세균확산지전신。세균계수결과현시，정조어감염적세균량수시간연장불단증가，단감염후10 h침입어체세포중적균량현저저우8 h。단백면역인적결과현시，감염후8 h Lc3-Ⅱ표체현저승고，p62표체현저강저。투사전자현미경하，감염후8 h유어세포중가관찰도자서체화자서용매체결구。이상결과제시，용서상한사문균침염유년반마어구건적모형가용우추종병원균감염인기적이원자서적동태변화。
A Salmonella-zebrafish infection model was applied to study bacteria-induced xenophagy . Zebrafish larvae at 72 h post-fertilization were exposed to different doses of Salmonella typhimurium (S . typhimurium) .The survival curve of larvae was used to determine the appropriate dose for initial infection . The in vivo distribution of the bacteria was monitored by following the green fluorescent protein (GFP)-tagged S . typhimurium in zebrafish larvae .The bacterial count ,the expression levels of autophagy markers Lc3-Ⅱ and p62 and the formation of autophagosome under the transmission electron microscope were followed .The results revealed that after an exposed infection by S .typhimurium at a dose of 1 × 109 CFU/ml ,the initial larvae survival rate was 100% in 2 d ,and the larvae began to die at day 3 .The bacteria invaded into the larvae within 4 h after infection and could be detected around the body in 3 d .Although the amount of bacteria in whole-larvae was increasing over time , the amount of intracellular bacteria was decreased more significantly at 10 h after infection than at 8 h .Western blotting analysis showed that Lc3-Ⅱ was significantly increased ,but p62 was reduced at 8 h after infection .Under the transmission electron microscope , autophagosome and autolysosome were observed in larvae at 8 h after infection . The preliminary results indicate that Salmonella-zebrafish model can be used to track the dynamic changes in autophagy induced by pathogens .