补肾中药对青春期大鼠下丘脑生长抑素基因及其蛋白表达的影响(英文)

Effects of kidney-nourishing Chinese drug on hypothalamus somatostatin gene and its protein expression of rats in puberty

背景:性早熟患儿在下丘脑-垂体-性腺轴提前启动的同时伴有骨骼生长加速,骨骺提前融合,临床上在发病时采用滋阴泻火中药可延缓骨龄提前融合,到性发育年龄时改用益肾填精中药,可促进性发育及促进骨骼生长加速,性早熟患者性发育及骨生长过程与正常青春期发育相同,只是时间的提前。中药改变骨生长的机制及对影响骨生长的生长抑素的作用尚不明确。目的:探讨补肾中药对下丘脑室周核生长抑素的调节作用。设计:完全随机分组设计,对照实验。单位:上海交通大学附属儿童医院内分泌科。材料:实验于2001-10/2002-04在上海复旦大学附属儿科医院儿科研究所完成。采用清洁级纯种雌性SD大白鼠共24只。随机分为4组:对照Ⅰ组,滋阴泻火药组,对照Ⅱ组,滋阴泻火+益肾填精药组,每组6只。方法:①对照Ⅰ组:灌胃生理盐水5mL/次,1次/d,共1个月;滋阴泻火药组:灌胃滋阴泻火中药合剂(生地黄、炙龟板、黄柏、知母等),5mL/次,1次/d,共1个月;对照Ⅱ组:灌胃生理盐水5mL/次,1次/d,共2个月;滋阴泻火+益肾填精药组:先灌胃滋阴泻火中药5mL/次,1次/d,1个月,再喂饲益肾填精中药合剂(熟地黄、龟板胶、仙灵脾、鹿角胶等),5mL/次,1次/d,1个月。②采用漂浮法原位杂交、常规免疫组化技术。以医学图像分析软件,作各组大鼠下丘脑室周核生长抑素mRNA、生长抑素免疫反应阳性神经元计数并检测其阳性细胞面积吸光度(A值)反映其基因表达及蛋白表达的水平。单位面积阳性神经元数即每平方毫米面积中所含阳性神经元数量。③计量资料比较采用单因素方差分析处理,组间差异采用F检验。主要观察指标:滋阴泻火及益肾填精中药对下丘脑室周核生长抑素基因和蛋白表达水平的影响。结果:大鼠24只均进入结果分析。①下丘脑室周核生长抑素mRNA阳性神经元数、单位面积阳性神经元数和阳性细胞面积A值:滋阴泻火药组明显高于对照Ⅰ组(P<0.05),滋阴泻火+益肾填精药组明显低于滋阴泻火药组(P<0.05)。②滋阴泻火药组下丘脑室周核生长抑素阳性神经元数、单位面积阳性神经元数、阳性细胞面积A值:滋阴泻火药组明显高于对照Ⅰ组(P<0.05),滋阴泻火+益肾填精药组明显低于滋阴泻火药组(P<0.05)。结论:滋阴泻火与益肾填精中药可通过对下丘脑生长抑素基因表达与蛋白表达的调控,调节垂体生长激素的合成及释放。

BACKGROUND： In association with advanced startover of hypothalamuspituitary-sexual gland axis, the growth of skeleton is accelerated and epiphyseal fusion occurred earlier in puberty precocity children. In clinic, Chinese drug for nourishing yin and reducing fire retards bone age and the advanced fusion in the disease. At the age of sexual development, Chinese drug for benefiting kidney to supplement essence is replaced to promote sexual development and accelerate skeleton growth. The patients with puberty precocity are the same to that in normal puberty in sexual development and bone growing, just being advanced in time. But, it is still unknown the mechanism of Chinese drugs on bone growth and effects on somatostatin （SS） in hone growth. OBJECTIVE： To explore the regulation of kidney-nournishing Chinese drug on periventricular nucleus （PeVN） SS. DESIGN： Completely randomized group division and control experiment were designed. SETTING： Department of Endocrinology, Children＇s Hospital Affiliated to Shanghai Jiantong University MATERIALS： The experiment was performed in Institute of Pediatrics of Children＇s Hospital Affiliated to Shanghai Fudan University from October 2001 to April 2002, in which, 24 SD white rats were employed, of purebred, female and clean grade. 4 groups were randomized, named the control A, the group with drugs of nourishing yin and reducing fire （experiment group No.1）, the control B and group with drugs of nourishing yin and reducing fire ＋ benefiting kidney to supplement essence （experiment group No.2）, 6 rats in each group. METHODS： ① Control A： physiological saline Was applied for gastric perfusion, 5 mL each time, once per day, totally for 1 month. Experiment No,1 group： Chinese mixture for nourishing yin and reducing fire was applied for gastric perfusion, composed of shengdihuang （Radix Rehmannlae）, zhiguiban （Chinemys reevesii.）, huangba/（Cortex Phellodendri） and zhimu （Rhizoma Anemarrhenae） etc, 5 mL each time, once per day, totally for 1 month. Control B： physiological saline was applied for gastric perfusion, 5 mL each time, once per day, totally for 2 months. Experiment No.2 group：Firstly, Chinese mixture for nourishing yin and reducing fire was applied for gastric perfusion, 5 mL each time, once per day, totally for 1 month. And then Chinese mixture for benefiting kidney to supplement essence was applied for breeding, composed of shudihuang （Radix Rehmanniae Preparata）, guibanjiao （Colla Carapacis Testudinis）, xianlingpi, lujiaojiao （Colla Comus Cervi） etc., 5 mL each time, once per day, totally for 1 month. ② In situ hybridization staining procedure with floating and routechnique. Medical imaging analysis software was used to assay PeVN SS mRNA, SS immunoreactive neuron counts and absorption （A value） of positive cell area that reflects the gene expression and protein expression. Number of positive neurons of unit area refers to the positive neuron counts in each square millimeter. ③Singer-factor analysis of variance was used for the comparison of measurement data. F test was adopted for difference comparison among groups. MAIN OUTCOME MEASURES： Effects of Chinese drugs for nourishing fin and reducing fire and benefiting kidney to supplement essence on PeVN SS gene mRNA and protein expression RESULTS： 24 rats were all in result analysis. ① PeVN SS mRNA positive neuron counts, positive neuron counts of unit area and A value of positive cell area： Those in experiment No.1 group was higher remarkably than the those of control A （P 〈 0.05）; and those in experiment No.2 group were lower remarkably than those of experiment No. 1 group （P 〈 0.05）. ② PeVN SS positive neuron counts, positive neuron counts of unit area and A value of positive cell area ： those in experiment No.1 group： those in experiment No.1 group were higher remarkably than those of control A （P 〈 0.05） and those in experiment No.2 group were lower remarkably than those of experiment No.1 group （P 〈 0.05）. CONCLUSION： By controlling hypothalamus SS gene expression and protein expression, Chinese drugs for nourishing yin and reducing fire and benefiting kidney to supplement essence regulate the synthesis and releasing of pituitary growth hormone.