碘缺乏和甲状腺功能减退对大鼠仔鼠小脑细胞外信号调节激酶1／2蛋白表达的影响

Effects of iodine deficient and hypothyroid on protein expression of extracellular signal-regulate kinase （ERK1/2） in the cerebellum of rats

目的观察碘缺乏和甲状腺功能减退（甲减）对大鼠仔鼠小脑细胞外信号调节激酶1／2（extracellular signal—regulate kinase，ERK1／2）蛋白表达的影响。方法健康Wistar大鼠28只，雌性，60日龄。将大鼠按体质量随机分为对照组、碘缺乏病组、甲减组，甲减组根据饮水中含丙基硫尿嘧啶（PrrU）剂量分为5mg／L组和15mg／L组。每组各7只。母鼠妊娠后，分别选用低碘饲料及PrrU饮水诱导建立低碘及甲减大鼠动物模型。仔鼠出生7、14、21、28、42d时分别处死，测定仔鼠小脑质量；取小脑组织进行镀银染色和免疫组织化学链霉菌抗生物素蛋白-过氧化物酶（S—P）染色，光镜下观察小脑形态学变化，图像分析仪下观察ERK1／2蛋白表达。结果仔鼠出生14、21、28、42d时，小脑质量各组间比较差异有统计学意义（F=6．325、8．870、16．191、21．574，P均〈0．05）；碘缺乏病组仔鼠小脑质量[（0．0945±0．0233）、（0．1347±0．0046）、（0．1542±0．0094）、（0．1949±0．0048）g]明显低于对照组[（0．1856±0．0123）、（0．2049±0．0098）、（0．2268±0．0065）、（0．2606±0．0086）g，P均〈0．05]和甲减组[5mg／L组：（0．1741±0．0172）、（0．1927±0．0103）、（0．2181±0．0064）、（0．2583±0．0054）g，P均〈0．05；15mg／L组：（0．1604±0．0083）、（0．1682±0．0103）、（0．1996±0．0073）、（0．2579±0．0067）g，P均〈0．05]。出生7d时，对照组、甲减组仔鼠小脑皮质具有典型的层状结构，各层之间界限清晰，但碘缺乏病组各层之间界限模糊；出生21d时，与对照组比较，15mg／L组和碘缺乏病组仔鼠小脑外颗粒细胞消失延迟，仍有2～3层外颗粒细胞；出生28、42d时。甲减组和碘缺乏病组仔鼠小脑分子层厚度低于对照组。小脑组织ERK1／2平均积分光密度，在出生7d时，组间比较差异无统计学意义（F=1．102，P〉0．05）；在出生14、21、28、42d时，各组间比较差异有统计学意义（F=16．131、13．543、26．953、41．583，P均〈0．01）；碘缺乏病组（7．3245±0．5070）、（8．3606±1．0683）、（9．1217±1．0402）、（12．1587±0．7581）和甲减组[5mg／L组：（11．4307±15200）、（14．919±0．8497）、（16．0082±1．1130）、（15．7721±0．8293）；15mg／L组：（7．8538±0．9775）、（11．2461±0．8138）、（12．7800±1．3783）、（13．0871±1．1450）]明显低于对照组[（16．2831±0．5143）、（20．2653±0．9551）、（22．7485±1．0267）、（22．1725±0．9939），P均〈0．01]。结论碘缺乏和甲减可使仔鼠小脑产生明显的形态学改变，降低大鼠仔鼠小脑组织ERK1／2蛋白表达，影响神经系统的发育。

Objective To study the effects of iodine deficiency and hypothyroidism on protein expression of extracellular signal-regulate kinase （ERK1/2 ） in the cerebellum of rats. Methods Twenty-eight healthy Wistar rats, female, 60 days old, were randomly divided according to their body weight into control group, iodine deficient group and hypothyroidism groups. Hypothyroidism groups in accordance with drinking water containing propyhhiouracil （PTU） were divided into doses of 5 mg/L and 15 mg/L groups, 7 rats in each group. Rats after pregnancy, iodine deficient rats were administered with iodine-deficient diet and hypothyroid rats were administered with PTU in drinking water. Pup＇s cerebellum in each group were weighed on day 14,21,28 and 42. Cerebellum tissue was observed for cerebellar morphology using silver staining and detected for ERK1/2 protein using immunohistochemistry on day 7,14,21,28 and 42. Results On day 14,21,28 and 42, cerebellum weight of pups from iodine-deficient [ （0.0945 ± 0.0233）, （0.1347± 0.0046 ）, （ 0.1542 ± 0.0094 ）, （ 0.1949 ± 0.0048 ） g ] were significantly lighter than control [ （0.1856 ± 0.0123）, （0.2049 ± 0.0098）, （0.2268 ± 0.0065）, （0.2606 ±0.0086）g, all P 〈 0.05] and hypothyroidism groups [for 5 mg/L group： （0.1741 ± 0.0172）, （0.1927± 0.0103）, （0.2181± 0.0064）, （0.2583± 0.0054）g, all P 〈 0.05; for 15 mg/L group： （0.1604 ±0.0083）, （0.1682 ± 0.0103）, （0.1996 ± 0.0073） and （0.2579 ± 0.0067）g, all P 〈 0.05] the difference had statistical signifieance（F = 6.325,8.870,16.191 and 21.574, all P 〈 0.05）. Compared to the controls on day 7, iodine-deficient group didn＇t have clear layers; on day 21, disappearance of external granule cells from iodine-deficient and 15 mg/L groups was delayed, still two or three layer external granule cells remained; on day 28 and 42, molecular layer from 5, 15 mg/L and iodine-deficient groups and became thinner. Immunohistochemistry showed that on day 7, there was no statistical difference of integrated optical density average of ERK1/2 , in all the groups（F = 1.102, P 〉 0.05）; on day 14,21,28 and 42, integrated optical density average of ERK1/2 in iodinedeficient group[ （7.3245 ± 0.5070）, （8.3606 ± 1.0683）, （9.1217 ±1.0402）, （12.1587 ± 0.7581）, all P 〈 0.01 ] and hypothyroidism groups[for 5 mg/L group：（11.4307 ±1.5200）, （14.919 ±0.8497）, （16.0082± 1.1130）, （15.7721 ± 0.8293）, all P〈 0.01; for 15 mg/L group： （7.8538 ± 0.9775）, （11.2461 ± 0.8138）,（12.78 ± 1.3783）, （13.0871 ± 1.1450）, all P 〈 0.01 ] was significantly lower than those of controls[ （16.2831 ± 0.5143）, （20.2653± 0.9551 ）, （22.7485 ± 1.0267）, （22.1725 ± 0.9939）, all P 〈 0.01 ], the difference having a statistical siguifieance（F = 16.131,13.543,26.953,41.583, all P 〈 0.01）. Conclusions Iodine deficiency and hypothyroidism during critical periods of brain development may change cerebellar morphology and down regulate the protein expression of ERK1/2, which may result in damage of cerebellum development.