秀丽隐杆线虫识别辐射损伤小鼠尿液的研究

Identification of radiation injury mouse urine by Caenorhabditis elegans

目的研究秀丽隐杆线虫(简称线虫)对照射后小鼠尿液的趋向性及与照射剂量和时间的关系。方法按照区组随机法将168只C57/BL6小鼠分为对照组和照射组,对照组为未照射的小鼠;照射组为使用137Csγ射线进行全身照射后的小鼠,剂量率为0.84 Gy/min。(1)照射后不同时间点的爬行实验:将小鼠分为7.5 Gy照射组和对照组,每组12只,于照射后0、2、4、8、12、24、48、72 h收集尿液;(2)不同剂量照射后的爬行实验:将小鼠分为2、4、6 Gy照射组和对照组,每组12只,于照射后24 h收集尿液。(3)时间与剂量之间的正交实验:将小鼠分为2、4、6 Gy照射组和对照组,每组12只,于照射后4、8、24 h收集尿液。(4)风扇实验和加热实验:将小鼠分为7.5 Gy照射组和对照组,每组12只,于照射后24 h收集尿液,分别将尿液用风扇吹风和加热后进行爬行实验。线虫培养至L3幼虫期,将其分别和每组小鼠的尿液滴至培养皿的不同区域,于体视显微镜下观察并计算每个区域中线虫的百分数。2组间的比较采用独立样本t检验和配对t检验。结果(1)照射后不同时间点的爬行实验:7.5 Gy照射后8 h时线虫开始出现聚集,与对照组相比,照射组在照射后8、12、24、48、72 h时线虫的百分数差异均有统计学意义(t=4.073~67.518,均P<0.01)。(2)不同剂量照射后的爬行实验:与对照组相比,2、4、6 Gy照射组区域线虫的百分数均增加[(29.33±5.79)%对(69.58±7.00)%、(11.58±3.60)%对(84.42±5.55)%、(9.08±2.19)%对(88.58±3.45)%],且差异均有统计学意义(t=11.955、30.320、51.463,均P<0.001)。(3)时间与剂量之间的正交实验:在照射后8、24 h,2、4、6 Gy照射组小鼠尿液的线虫百分数与对照组相比,差异均有统计学意义(t=17.628~133.349,均P<0.001)。(4)风扇实验和加热实验:风扇实验结果显示,照射组和对照组的线虫百分数均增多,但二者的差异无统计学意义(t=1.250,P>0.05);加热实验结果显示,小鼠尿液加热前后整体重量的差异有统计学意义[(1.060±0.028)g对(1.051±0.026)g,t=11.814,P<0.001],照射组和对照组的线虫百分数均增多,但二者的差异无统计学意义(t=0.585,P>0.05)。结论线虫对照射后小鼠尿液中挥发性代谢物具有趋向性,且可辨别低剂量照射。

Objective To study the relationship between urine tendency of Caenorhabditis elegans(C.elegans)to irradiated mice and administration dose and time of radiation.Methods A total of 168 C57/BL6 mice were divided into control group and irradiation group by randomized block design.The irradiation group was treated with 137Csγtotal body irradiation with 0.84 Gy/min.Mice and wild-type C.elegans were used to perform experiments with the following treatments:(1)crawling test at different time points after irradiation;the mice were divided into 7.5 Gy irradiation group and control group(n=12).After 7.5 Gy total body irradiation,urine samples were collected at 0,2,4,8,24,48,and 72 h.(2)Crawling test after different doses of irradiation;the mice were divided into 2,4,and 6 Gy irradiation groups and control groups(n=12).Urine samples were collected at 24 h after irradiation.(3)Orthogonal experiment between irradiation time and dose;the mice was divided into 2,4,and 6 Gy irradiation groups and control groups(n=12),and urine samples were collected at 4,8,and 24 h after irradiation.(4)Fan test and heating test;the urine samples were collected from 7.5 Gy total body irradiation mice after 24 h,accompanied by a blow on one side of the petri dish or heating of the urine prior to crawling test.C.elegans were cultured to L3 larval stage,and the urine of each group was dripped to different areas of the culture dish.The percentage of C.elegans in each area was observed and calculated under stereomicroscope.Independent-sample t test and paired t test were used for comparison between groups.Results(1)Crawling test at different time points after irradiation:C.elegans began to gather at 8 h after 7.5 Gy irradiation.Compared with the control group,significant differences in the percentage of C.elegans were observed at 8,12,24,48,and 72 h after irradiation in the irradiation group(t=4.073-67.518,all P<0.01).(2)Crawling test after different doses of irradiation:compared with the control group,the percentages of C.elegans in the urine area of the 2,4,and 6 Gy irradiation groups were increased((69.58±7.00)%,(84.42±5.55)%,and(88.58±3.45)%,respectively,and(29.33±5.79)%,(11.58±3.60)%,and(9.08±2.19)%in the control group),and the differences were statistically significant(t=11.955,30.320,51.463;all P<0.001).(3)Orthogonal experiment between irradiation time and dose:compared with the control group,the percentages of C.elegans in the urine of mice showed significant differences at 8,24 h after 2,4,and 6 Gy irradiation(t=17.628-133.349,all P<0.001).(4)After the fan test,approximately(3.17±1.37)%C.elegans were distributed in the irradiation group and(2.38±1.26)%in the control group,without significant difference(t=1.250,P>0.05).The results of heating test showed a significant difference in the overall weight of urine before and after heating((1.060±0.028)g vs.(1.051±0.026)g,t=11.814,P<0.001).Approximately(17.46±11.00)%C.elegans were distributed in the irradiation group and(12.70±9.91)%in the control group,but no significant difference was found between the two groups(t=0.585,P>0.05).Conclusion C.elegans have a tendency of volatile metabolites in urine of irradiated mice and can distinguish low-dose irradiation.