神经管畸形DNA损伤单细胞凝胶电泳检测

Detection on DNA damage by single cell gel electrophoresis assay technology

目的利用单细胞凝胶电泳技术检测大鼠神经管畸形发生过程中DNA损伤及影响因素。方法Wistar孕鼠20只,按体重随机分为神经管畸形模型组和生理盐水对照组。模型组于孕13 d一次性腹腔注射环磷酰胺12.5mg/(kg·bw),正常对照组于孕13d给予0.3 ml的生理盐水。孕14 d时各组随机处死2只孕鼠,每只孕鼠各取3只胎鼠的脑组织,利用单细胞凝胶电泳技术检测神经管畸形发生过程中胚胎神经细胞DNA损伤情况并分析影响因素。孕20 d时处死各组动物,检测动物的生长发育情况以及各组的畸形发生率。结果正常对照组的胎鼠神经细胞呈现典型的圆形,而模型组出现特征性彗星形态,即很小的彗星头,大而圆的彗星尾,且彗星尾长[(16.35±5.59)μm]明显高于对照组[(7.28±1.76)μm]。于孕20 d时处死各组动物,发现模型组动物胎鼠的生长发育指标包括身长、体重和尾长均明显小于对照组(P<0.05),畸形发生率(67%)明显高于对照组(P<0.05)。细胞悬液和胶的浓度、裂解及荧光染色的时间是影响实验的因素。结论单细胞凝胶电泳技术可以检测神经管畸形发生时胚胎神经细胞中DNA损伤情况,注意实验的影响因素可增加该法的特异性和灵敏度。

Objective To study the detection on DNA damage by single cell gel clectrophoresis assay technology during rats＇ neural tube defect process. Methods Twenty pregnant rats were randomly divided into control group and NTDs model group according to their weight. On the 13 d of gestation, the pregnant rats of the model group were given cyclophsphamide 12.5 rag/（kg· bw） via intraperitoneal administration, the control group was given 0.3 ml N. S in the same way. On the 14 d of gestation, two rats of each group were executed. The DNA damage of three embryo＇s brain tissue in each pregnant rat was checked through single - cell gel electrophoresis technology. Furthermore, influence factors of SCGE were analyzed. Results Comet cells were found in the model group but were not found in control group. The comet cells showed small head and big tail. Tail length of comet cells in model group [ （16.35 ± 5.59）btm] was longer significantly than that of neuron cells in control group [ （7.28 ± 1.76）μm]. Ratio of abnormity in model group（67 % ） was significantly increased（ P 〈 0.05） and the developmental index was significantly decreased in model group（ P 〈 0.05）. There were some factors affecting the experiment including concentration of cell suspension and gel, time of split as well as fluorescent staining. Conclusion DNA damage in NTDs can be checked by single cell gel electrophoresis assay technology. Besides, some details should be emphasize in order to improve the sensitivity and specificity.