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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。
Objective To explore the optimal method for inactivating Escherichia coli OP50 feeding nematode and its suitable concentration in liquid culture system when the index of thermal tolerance of nematodes was taken as test endpoint. Then, the effects of PM 2.5 on the heat tolerance of nematodes were evaluated by optimized feeding method, which lays the foundation for the study on the effects of PM 2.5 on the life span of nematodes and their molecular mechanisms. Methods Two kinds of common inactivation methods (heat treatment and cobalt 60 irradiation) were used to treat Escherichia coli OP50 strain, and untreated live E. coli OP50 strain (control group) were fed nematode, respectively, and each group of nematode liquid medium (S-medium solution) to bacterial concentrate (A = 1.949) were 1:50 and 1: 100, respectively. By observing the basic morphological differences of the growth and development of nematode fed with different treatments and the effect of heat tolerance on the 5th day after feeding, the differences between the heat treatment, the cobalt 60 irradiation treatment and the control live bacteria were evaluated Determine the inactivation and appropriate concentrations of E. coli OP50 bacteria that do not affect the growth and development of nematodes and no toxic effects. On this basis, the water-soluble PM_ (2.5) samples were collected from winter in Beijing (94.587μg / ml in group A and 119.29μg / ml in group B) Sexual effects. Results The nematodes fed by the heat-treated bacteria group had shorter body length, narrower body width, slower growth rate than the control group, and no difference between the control group and the growth status of the nematodes fed the cobalt-60 irradiation group . At the same time, it can be seen that when the proportion of Escherichia coli OP50 in liquid medium is 1: 100, the growth of nematodes is slower than that of 1:50. At the 10th day of exposure, the survival curve of C. elegans could be shifted to the left by the group of positive longevity (2% glucose) and PM 2.5 (2.5) under heat stress. Compared with the blank control group, the average survival time of the positive longevity group was shortened by 48.43% (P <0.01), and the average survival time of the PM 2.5 treated group was shortened by 26.29% (group A) and 31.25% (group B) ) (P <0.01). Conclusion Co - 60 irradiation is a suitable method for inactivating Escherichia coli OP50 in the experimental study on heat tolerance of nematodes. The volume ratio of bacterial concentrate to liquid medium is 1:50, which is better than 1: 100. Under the action of PM 2.5, The average 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 and may further affect the life span of nematodes.