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随着核技术的发展和核能应用的增多,核泄漏或核战争发生的可能性增加,一旦事故发生,人们将受到电离辐射造成的不可逆损伤。核工业的从业人员也存在潜在风险,若意外暴露于电离辐射下,轻者可导致机体发生病理变化,重者会损伤其组织和器官[1-2]。核在医疗中的应用越来越多,比如放疗是癌症患者常用的治疗方法。其射线产生的不良反应包括急性胃肠道反应、皮肤反应,程度从轻微皮疹、水肿、腹泻、呕吐到严重溃疡、组织器官的坏死、瘘及其他并发症的发生[3]。约85%接受放疗的患者会出现中度至重度的不良反应,但是临床上没有用于评价放射性不良反应严重程度及预后的有效指标。早期准确诊断辐射损伤是目前临床上急需的。然而,利用稳定的染色体畸变进行辐射损伤诊断的生物剂量法费时费力,且有许多局限性,不适合对受照个体进行快速地判断和分类[4-5]。
秀丽隐杆线虫(Caenorhabditis elegans,简称线虫)是第一个基因组被完全测序的多细胞生物[6]。目前的研究数据表明,38%以上的线虫蛋白表达基因与人类同源,60%~80%的人类基因都可在线虫基因组中找到同源基因,其中包括40%的人类疾病相关基因[7]。因此,线虫作为一种模式生物已被广泛应用于人类疾病的研究。线虫独特的神经元结构决定了它具有灵敏的嗅觉[8-10]。有研究结果表明,野生型线虫对人类肿瘤细胞的分泌物、肿瘤组织以及肿瘤患者的尿液具有趋向性,但对健康者的尿液没有趋向性,同时也发现,线虫能通过嗅觉神经元“感觉”到尿液中的气味[11-12]。这种趋向性已在乳腺癌、宫颈癌和黑色素瘤等多种类型肿瘤中得到证实[13]。由此可见,通过嗅觉灵敏的线虫对患者的体液气味进行分析,可以更好地观察和预测疾病的发生发展[14-18]。
本研究通过全身照射建立小鼠辐射损伤模型,利用线虫的嗅觉,研究其对照射后小鼠和未照射小鼠的尿液是否表现出不同的趋向性、照射剂量和照射后不同时间的关系、照射后小鼠的尿液中是否有吸引线虫的代谢性物质等,为临床辐射损伤的诊断和治疗提供新思路,为辐射损伤代谢标志物的研究打基础,以期开发出一种用于临床的放射性核素造成不良反应的评估体系。
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趋化实验结果见图2A,结果显示线虫对照射后小鼠的尿液有明显趋向性,与对照组相比,照射组区域内聚集了更多的线虫。0.5 h后,对照组有约(1.83±0.17)%的线虫,而照射组线虫达(14.17±1.01)%,二者的差异有统计学意义(P=0.002,图2B)。
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如图3所示,7.5 Gy照射后8 h时线虫开始出现聚集,这说明此时小鼠尿液中产生了相关代谢物,且该代谢物在照射后72 h并未消失,同样可吸引线虫在尿液区域内聚集。与对照组线虫的百分数相比,照射组在照射后8、12、24、48、72 h时的差异均有统计学意义(t=4.073、42.947、53.333、67.518、61.250,均P<0.01)。
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如图4所示,2 Gy照射后小鼠的尿液可吸引线虫聚集,照射组区域的线虫百分数为(69.58±7.00)%、对照组为(29.33±5.79)%。同样,4、6 Gy照射组也有线虫聚集[照射组线虫的百分数分别为(84.42±5.55)%和(88.58±3.45)%、对照组分别为(11.58±3.60)%和(9.08±2.19)%],与对照组相比,经不同剂量照射后小鼠的尿液中线虫的百分数均增加,且差异均有统计学意义(均P<0.001)。由此可知,线虫灵敏的嗅觉可识别2 Gy及以上剂量照射后小鼠的尿液。
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由表1可知,在2、4、6 Gy不同剂量照射4 h后,小鼠尿液中无相关代谢物,其线虫的百分数与对照组相比,差异无统计学意义(t=2.152、−1.012、−0.087,均P>0.05);在照射后8 h,不同剂量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(均P<0.001)。这表明在照射后8 h时,不论照射剂量多少,该代谢途径已经产生。
组别 4 h 8 h 24 h 对照组(n=12) 20.00±2.04 21.00±1.41 20.17±1.75 2 Gy照射组(n=12) 22.25±2.99 63.25±7.55a 83.92±4.27a 4 Gy照射组(n=12) 18.58±3.26 77.33±8.77a 88.50±3.34a 6 Gy照射组(n=12) 19.92±1.97 72.58±6.14a 88.75±1.76a 注:a表示与对照组相比,差异均有统计学意义(t=17.628~133.349,均P<0.001) 表 1 不同剂量照射后不同时间点小鼠尿液中秀丽隐杆 线虫的百分数(
±s,%)$\bar x $ Table 1. Percentage of Caenorhabditis elegans in urine of mice at different time points after different doses of irradiation (
±s, %)$\bar x $ -
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[(3.17±1.37)%对(2.38±1.26)%,t=1.250,P>0.05],即风扇实验后线虫不能识别照射后小鼠的尿液而产生聚集。加热实验结果显示,小鼠尿液加热前后整体重量差异有统计学意义[(1.060±0.028)g对(1.051±0.026)g,t=−11.814,P<0.001],这表示有大量组分挥发;蒸发后尿液的线虫爬行结果显示,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[(17.46±11.00)%对(12.70±9.91)%,t=0.585,P>0.05],即经加热后的小鼠尿液并不能被线虫识别出是否经过照射。因此,我们认为照射后小鼠尿液中能被线虫特异性识别的物质为挥发性代谢物。
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
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摘要:
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137Cs γ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。 Abstract: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 137Cs γ total 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. -
Key words:
- Caenorhabditis elegans /
- Radiation injuries /
- Unrine tendency /
- Volatile metabolite
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表 1 不同剂量照射后不同时间点小鼠尿液中秀丽隐杆 线虫的百分数(
±s,%)$\bar x $ Table 1. Percentage of Caenorhabditis elegans in urine of mice at different time points after different doses of irradiation (
±s, %)$\bar x $ 组别 4 h 8 h 24 h 对照组(n=12) 20.00±2.04 21.00±1.41 20.17±1.75 2 Gy照射组(n=12) 22.25±2.99 63.25±7.55a 83.92±4.27a 4 Gy照射组(n=12) 18.58±3.26 77.33±8.77a 88.50±3.34a 6 Gy照射组(n=12) 19.92±1.97 72.58±6.14a 88.75±1.76a 注:a表示与对照组相比,差异均有统计学意义(t=17.628~133.349,均P<0.001) -
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