血管紧张素Ⅱ受体2在小鼠早期肾脏发育中的表达

Expression of angiotensinⅡsubtype 2 receptor in early renal development of mice

目的:观察血管紧张素Ⅱ受体2在小鼠早期肾脏发育中的表达及其与肾脏发育的关系。方法:实验于2006-06/10在辽宁医学院科学实验中心与组织胚胎学实验室完成。选取24只健康昆明小白鼠,雌、雄各半。按雌雄比例1∶1同窝饲养,采用检查阴道栓脱落的方法确定雌鼠受孕时间。以观察到阴道栓脱落的最早时间计为胚龄0d。分别取胚龄14,16,18d胎鼠和生后1,3,7d龄仔鼠肾脏,每组4只。孕鼠经乙醚麻醉后剖腹取出胎鼠,胚龄14,16,18d全肾固定。仔鼠经乙醚麻醉后,于腹后壁取出肾脏,用排刀法沿横轴将肾脏切开,入100g/L中性甲醛固定,系列乙醇脱水,二甲苯透明,石蜡定向包埋,做厚5μm连续切片。①将切片用血管紧张素Ⅱ受体2抗体进行染色,稀释度为1∶100,染色步骤按免疫组化试剂盒说明书进行。②采用体视学方法测定血管紧张素Ⅱ受体2在肾小球及肾小管处阳性细胞的体密度(VV=ΣPx/ΣPc),测算出各胚(d)龄小鼠各期皮质内的VV值,ΣPx为方格系统落在肾小体及肾小管内血管紧张素Ⅱ受体2阳性细胞的点数,ΣPc为方格系统落在参照系(相应的各期肾小体和肾皮质)的点数。③各组小鼠取右肾,加入4倍体积的裂解液,粉碎,离心收集上清,用免疫印记分析法测定总蛋白含量。结果:①血管紧张素Ⅱ受体2表达在生肾区的肾小体、输尿管芽及间充质,孕16d除在生肾区外开始出现在肾小管,且随着肾小体的逐渐成熟表达量降低。孕18d随着生肾区面积的减少表达量降低,而在肾小管的表达量有所增加;生后随着生肾区的进一步减小,表达量进一步降低,到生后7d时只有微量表达,生后1d在肾小管表达减少,生后3d没有表达。②血管紧张素Ⅱ受体2在孕16d时开始表达在肾小管,阳性细胞体密度为(11.17±2.01)%,孕18d达到(19.19±3.46)%,生后1d为(5.18±1.56)%。生后3d没有表达。Ⅰ期肾小体内血管紧张素Ⅱ受体2的阳性细胞体密度为(57.88±8.77)%。Ⅱ期血管紧张素Ⅱ受体2阳性细胞体密度为(51.47±5.26)%,进入Ⅲ期肾小体时,血管紧张素Ⅱ受体2阳性表达明显减少(22.41±3.46)%,Ⅳ期肾小体内(17.17±3.09)%,Ⅴ期肾小体血管紧张素Ⅱ受体2阳性表达基本消失(6.28±2.80)%。③血管紧张素Ⅱ受体2蛋白表达随着胚龄的增加含量逐渐降低,生后降低明显。结论:血管紧张素Ⅱ受体2在胚胎期大量表达在生肾区的输尿管芽和肾小体,在间充质及肾小管也有表达,生后表达量迅速降低,说明血管紧张素Ⅱ受体2对肾脏各结构的发育起着非常重要的作用。

AIM： To investigate the expression of angiotensin Ⅱ subtype 2 receptor in the developing renal as well as the association with renal development. METHODS： The experiment was performed in the Scientific Experiment Center and Department of Histology and Embryology, Liaoning Medical University from June to October 2006. Twenty-four healthy Kunming mica were selected with either sex. The mice were bred according to two genders at the ratio of 1：1. Conception time was determined by the shedding of pessary. The eadiest time that the pessary fell off was regarded as embryonic 0 day （E0）. The kidney of the foetus mica of 14, 16, and 18 days and postnatal 1, 3, 7 days （N1） mice were taken with 4 mica in each group. The fetus mica were taken after the pregnant mica were etherized, and the whole kidney of E14, E16, and E18 mica were fixed. The kidney were moved from posterior abdominal wall of suckling mice after anesthesia with ether, then cut horizontally, and fixed with 100 g/L neutral formalin; the specimens were dehydrated with gradient alcohol, made transparent by dimethylbenzene, and embedded in paraffin routinely to prepare serial sections of 51μm.①According to the immunohistochemical kid, the sections were stained with angiotensin Ⅱ recapter 2 antibody at dilution of 1：100. ②The bulk density of positive cells in glomerulus and tubules was detected by stereology （Vv=∑ Px/∑Pc）. Vv value of cortex of each group was detected; ∑Px was the points of angiotensin Ⅱ subtype 2 receptor positive cells in glomerulus and tubules showed in the square system; ∑ Pc was the points at the corresponding reference strain. ③Right kidney of each group were obtained, and lysate of quadruple volume were added; the supematant was obtained after pulverization, and centrifuge; the total protein was measured by Western blot. RESULTS： ① Angiotensin Ⅱ receptor 2 was expressed in glomcrulus, ureteric bud and mesenchymal tissue of nephrogenic zone. At E16, the expression was found in tubules besides the nephrogenic zone, which decreased with the gradual maturation of glomcrulus. At E18, the expression was decreased with the reducing of nephrogenic zone, but increased in tubules; the expression was decreased with the reducing of nephrogenic zone after birth, and at N7, angiotensin Ⅱ receptor 2 was only slightly expressed. It was expressed little at N1 and no expression was found at N3. ②Angiotensin Ⅱ receptor 2 was expressed in glomcrulus at E16, and the density of positive calls was （11.17±2.01 ）%, which increased to （19.19±3.46）% at E18, but （5.18±1.56）% at N1. No expression was found at N3. The density of positive calls in renal corpuscle [ was （57.88±8.77）%; in renal corpuscle Ⅱ, it was （51.47±5.26）%, and in renal corpuscle In, it was obviously decreased （22.41±3.46）%,it was （17.17±3.09） % in renal corpuscle Ⅳ; in renal corpuscleV, it nearly disappeared, about （6.28±2.80）%. ③The expression of angiotensin Ⅱ receptor 2 protein decreased gradually with embryonic age and decreased sharply after birth. CONCLUSION： The expression of angiotensin Ⅱ receptor 2 is abundance in the ureteric bud and glomcrulus in developing renal, and is also found in mesenchymal tissue and tubules, which decreases sharply after birth, suggesting that angiotensin Ⅱ receptor 2 plays an important role in renal development.