镉染毒大鼠胚胎神经上皮中Zic2蛋白的表达

Cadmium-induced Changes in Zic2 Protein Expression Level in Neuroepithelial of Rat Embryos

目的探讨在神经系统发育过程中镉对 Zic2表达的时空顺序的影响。方法将40只 Wistar 孕鼠随机分为对照组和镉染毒组,镉染毒组孕鼠于胚胎6.5 d(embryonic day 6.5,E6.5)一次性按4 mg/kg 的剂量腹腔注射氯化镉,对照组和镉染毒组各分为4个亚组,分别在E9.0、E9.5、E10.5和 E11.5断椎处死孕鼠。随机取1/2妊娠子宫,在体视显微镜下取出胚胎,对胚胎神经系统分化进行评分。其余的1/2妊娠子宫置于4%多聚甲醛中固定,常规石蜡包埋,从胚体头端向尾端行5μm连续切片,采用二步法免疫组织化学技术检测 Zic2在神经上皮的表达情况。结果镉染毒组大鼠胚胎的神经系统发育异常,主要表现为发育迟缓、前后神经孔延迟闭合或不闭合等。镉染毒组大鼠胚胎的前脑、中脑和后脑的发育评分均低于相应的对照组,除 E9.5的中脑的发育评分与对照组相比,差异无统计学意义(P>0.05)外,其余差异均有统计学意义(9<0.05)。E9.5、E10.0、E10.5和 E11.5的镉染毒组的神经系统发育评分低于相应的对照组,差异均有统计学意义(t 值分别为2.71、4.72、3.78、5.61,P 值均小于0.05)。与对照组的大鼠胚胎相比较,镉染毒组 Zic2蛋白在 E9.5的神经板区的表达减少(t=1.978,P<0.05);在 E10.0的神经沟处表达明显减少(t=4.106,P<0.05),神经褶处有少量表达(t=5.677,P<0.05);在E10.5的神经管上表达明显减少(t=5.231,P<0.05);在 E11.5的前、中、后脑及神经管上主要表达在背侧,但是表达量明显低于对照组(t=3.405,P<0.05)。结论镉可以通过影响 Zic2表达的时空顺序,使大鼠胚胎神经系统发育异常。

Objective To study the effect of cadmium on the spatiotemporal sequence of Zic2 expression in the development of the nervous system. Methods Wistar rats were used in the initial portion of the study. Male and female animals were housed together overnight; the presence of sperms by vaginal smearing methods the following morning indicated that copulation had occurred, and that day was designated embryonic day 0.5 （E0.5）. Forty dams were divided randomly into the control group and the cadmium exposure group. The dams in the cadmium exposure group were treated with cadmium at a dose of 4mg/kg by intraperitoneal injection. The dams of control and cadmium exposure groups were divided 4 subgroups respectively, sacrificed at various times on embryonic days 9.0, 9.5. 10.5, and 11.5, laparotomized and the one half pregnant uterus were taken out. The embryos were taken out under stereomicroscope and the morphological score of the nervous system were calculated. The other pregnant uterus were fixed fxed with 4% paraformaldehyde routinely embedded in paraffin and eoronally sliced into slice 5μm of thickness for immunohistoehemical staining. Results Cadmium could lead to dysplasia of nervous system in embryos of rats. The main manifestations were growth retardation, delay closure or unelosures pre-and post-neuropores, and so on. Developmental scores of the forebrain, midbrain and hindbrain of rat embryos in the cadmium exposure groups were lower than the corresponding control groups, and in cadmium exposure groups had significant difference compared with those in control group （P〈0.05）, except the developmental score of midbrain in the E9.5 （P〉0.05）. According to developmental overall scores of nervous system, the scores of the cadmium exposure groups were lower than that of the control groups with significant difference on E9.5, E10.0, E10.5 and E 11.5（t=2.71, 4.72, 3.78. and 5.61 respectively, all P〈0.05 ）. Compared with the rats＇ embryos in the corresponding control groups, in the cadmium exposure groups Zic2 expression decreased in the midline region of the neural plate at E9.5 （t=1.978, P〈0.05）; at E10.0 Zic2 expression significantly decreased in neural grooves （t=4.106, P〈0.01） and a little expression in the neural folding （t= 5.677, P〈0.01）： at E10.5 Zic2 expression significantly decreased in the neural tube（t=5.231, P〈0.01）： at E11.5 Zic2 were expressed in the dorsal forebrain, midbrain and hindbrain, in the spinal cord, expression was restricted to the dorsal most region including the roof plate, but the amount of expression was lower than control group significantly （t=3.405, P〈0.01）. Conclusion Cadmium exposure may effect the spatial expression patterns of Zic2, make abnormal development of embryonic nervous system in rats.