【摘要】 目的本研究探索以线虫的热耐受性指标为检测终点时,喂饲线虫的食物大肠杆菌OP50菌株的最优灭活方法及其在液体培养体系中的适宜浓度。然后应用优化喂饲方法,评价PM_2.5对线虫热耐受性的影响,为进一步开展PM_2.5的线虫寿命影响及其分子机制研究奠定基础。方法采用2种常见灭活方法（热处理和钴60照射）处理大肠杆菌OP50菌株,及未处理的活大肠杆菌OP50菌株（设为对照组）分别喂饲线虫,设定每组线虫的液体培养基（S-medium溶液）与加入细菌浓缩液（A=1.949）的体积比分别为1∶50和1∶100 2个水平。通过观察不同处理菌喂饲下线虫生长发育的基本形态学差异及喂饲5 d后对热耐受性的影响来评价热处理、钴60照射处理菌组与对照活菌组之间的差异,从而确定不影响线虫生长发育且对毒性效应无干扰的大肠杆菌OP50菌的灭活方式及其适宜浓度。在此基础上,采用北京市冬季室外PM_2.5水溶性样品（终浓度分别为A组94.587μg/ml、B组119.29μg/ml）,处理线虫5和10 d后观察其对热耐受性的影响。结果热处理菌组喂饲的线虫体长较短,体宽较窄,生长较对照活菌组喂饲线虫缓慢,而钴60照射处理菌组喂饲的线虫生长状态和对照组差异无统计学意义。同时可见大肠杆菌OP50在液体培养基中所占比例为1∶100时线虫生长较1∶50时缓慢。染毒至第10天时,在热应激条件下阳性减寿组（2%葡萄糖）和PM_2.5染毒处理组均可使线虫生存曲线左移。与空白对照组相比阳性减寿组平均生存时间缩短了48.43%（P<0.01）,PM_2.5染毒处理组线虫平均生存时间分别缩短了26.29%（A组）和31.25%（B组）（P<0.01）。结论钴60照射是线虫热耐受性实验研究中较合适的大肠杆菌OP50灭活方法,细菌浓缩液和液体培养基的体积比为1∶50优于1∶100,PM_2.5作用下,线虫在热应激条件下平均生存时间缩短约1/3,提示PM_2.5降低线虫的热耐受能力,有可能进一步影响线虫的寿命。更多还原

【Abstract】 Objective The aim of this study was to explore the optimal inactivation method of Escherichia coli fed with nematodes and its suitable concentration in liquid culture system by detecting the heat tolerance index of nematodes as the end point. Then the optimized feeding method was used to evaluate the effect of PM_2.5 on the thermal tolerance of nematode,which laid the foundation for the further study on the effects of PM_2.5 on the life span and the molecular mechanism. Methods Nematode were fed with the two common inactivation method( heat treatment and ⁶⁰Co gamma irradiation treatment) of Escherichia coli OP50 strain treatment,and untreated live Escherichia coli OP50 strains（ control group）. Each group was set two levels of bacterial concentration,which were 1∶ 50 and 1∶ 100 volume ratio of liquid culture medium of nematode（ S-medium） and adding concentrated bacterial OP50（ A = 1. 949） respectively. By observing the morphological differences of the growth and development of nematodes with different treatments and the effects of the thermal tolerance of nematodes after 5 d feeding to evaluate the difference of heat treatment,⁶⁰Co gamma irradiation treatment group and control group,so as to determine the way to inactivate Escherichia coli OP50 and its suitable concentration does not affect the nematode growth and development without interference to the toxic effect. On this basis,using the outdoor PM_2.5 water-soluble samples in Beijing city in winter( final concentration of PM_2.5 was 94. 587 μg/ml in group A and 119. 29 μg/ml in group B),nematodes were treated for 5 and 10 d to observe its effect on the thermal tolerance of nematode. Result It showed that the body length of the heat treated bacteria was shorter and the body width was narrow,and the growth was slower than that of the control group,while the nematode growth status of the Co60 irradiation treatment group was not significantly different from that of the control group. At the same time,the proportion of OP50 in liquid medium was 1 ∶ 100 when the nematode growth was slower than 1∶ 50. Exposure to tenth days,in the heat stress conditions positive reduction group（ 2% glucose） and PM_2.5 treatment group can make the nematode survival curve left shift. Compared with the control group,the mean survival time of the positive life reduction group was shortened by 48. 43%（ P < 0. 01）,and mean survival time of the PM_2.5 treatment group was shortened by 26. 29%（ A group） and 31. 25%（ B group）（ P < 0. 01） respectively. Conclusion ⁶⁰Co irradiation is a suitable OP50 inactivation method in the study of thermal tolerance of nematodes. The volume ratio of bacterial concentrate and liquid medium was 1∶ 50 better than 1∶ 100. The result of heat tolerance test showed that under exposure of PM_2.5 ,the mean survival time of nematodes under heat stress was shortened by about 1/3,suggesting that PM_2.5 could reduce the heat tolerance of nematodes,which may further affect the longevity of nematodes.更多还原