摘要
【目的】研究神经激肽B(NKB)中枢给药对大鼠初情期启动、下丘脑Kisspeptin和促性腺激素释放激素(GnRH)基因及蛋白质的表达水平,以及血清中性腺激素含量的影响,以了解NKB在调节初情期方面的作用。【方法】将初情期前SD雌性大鼠分为NKB组、NKB拮抗剂组和对照组,分别侧脑室注射60 pmol/μL NKB溶液、0.3 nmol/μL NKB拮抗剂和生理盐水,注射剂量为10μL/只,每日观察阴门开启情况,统计阴门开启时间。所有大鼠均在阴门开启时采集血样,分离血清,并采集下丘脑、卵巢、子宫,称卵巢、子宫质量。分别采用免疫荧光共定位和实时荧光定量PCR(RT-qPCR)技术,分析下丘脑中GnRH和Kisspeptin蛋白和基因的表达情况,并分别通过酶联免疫吸附法(ELISA)和放射性免疫法(RIA)检测血清中NKB和性腺激素(雌二醇、孕酮)水平。【结果】NKB使大鼠阴门开启时间显著提前(P<0.05),卵巢质量显著降低(P<0.05),对子宫质量无明显影响(P>0.05);NKB拮抗剂使阴门开启时间显著推迟(P<0.05),对卵巢和子宫质量无显著影响(P>0.05)。与对照组相比,NKB组SD大鼠下丘脑Kiss1和GnRH mRNA的表达显著提高(P<0.05);GnRH+细胞数量在室旁核(PVN)和正中隆起(ME)显著增加(P<0.05),而在下丘脑弓状核(ARC)中显著减少(P<0.05),Kisspeptin+细胞数量在ARC和室周核(PeN)中显著增加(P<0.05),在PVN中无显著差异(P>0.05);血清中E 2和P 4水平均显著增加(P<0.05)。但在NKB拮抗剂组中,SD大鼠下丘脑Kiss1和GnRH mRNA的表达低于对照组,但无显著差异(P>0.05),GnRH+细胞数量在ARC中显著增加(P<0.05),在室旁核PVN和ME则显著降低(P<0.05),ARC和PeN中Kisspeptin+细胞的数量显著减少(P<0.05),且血清中E 2和P 4水平均显著降低(P<0.05)。【结论】NKB通过调节下丘脑Kisspeptin和GnRH的表达以及提高血清中E 2和P 4水平来促进大鼠初情期启动。
【Objective】This study investigated the effect of central administration of NKB on puberty onset,expression levels of Kisspeptin and gonadotrophin releasing hormone(GnRH)genes and proteins in the hypothalamus and concentration of gonadal hormone in serum of rats.【Method】Prepubertal Sprague-Dawley rats were randomly divided into NKB,NKB antagonist and control groups,and they were intracerebroventricularly injected with 60 pmol/μL NKB solution,0.3 nmol/μL NKB antagonist and normal saline at the dose of 10μL per rat,respectively.Then,the time of vaginal opening was recorded.Blood samples were collected when vulva opened and serum was separated.The hypothalamus,ovaries and uterus were collected,and the weight of ovaries and uterus were measured.The proteins and genes expressions of GnRH and Kisspeptin in hypothalamus were detected by fluorescence immunoassay and real-time quantitative PCR(RT-qPCR),respectively.The enzyme linked immunosorbent assay(ELISA)and radioimmunoassay(RIA)were used to analyze the concentration of NKB and serum gonadal hormones(E 2 and P 4).【Result】NKB significantly increased vaginal opening time of rats(P<0.05),significantly reduced ovary weight(P<0.05),and had no significant effect on uterine weight(P>0.05).NKB antagonist significantly delayed vaginal opening time(P<0.05),but had no significant effect on ovarian and uterine weights(P>0.05).In the NKB group,the expression of Kiss1 and GnRH mRNA in the hypothalamus was significantly increased(P<0.05).The number of GnRH+cells was significantly decreased(P<0.05)in arcuate nucleus(ARC)but was increased in paraventricularis nucleus(PVN)and median eminence(ME).The number of Kisspeptin+cells was significantly increased(P<0.05)in the ARC and periventricular nucleus(PeN)but there was no difference(P>0.05)in PVN.The concentrations of E 2 and P 4 in NKB group were significantly increased(P<0.05).However,in the NKB antagonist group,the expressions of Kiss1 and GnRH mRNA in hypothalamus were lower than those in the control group without significant difference(P>0.05).The number of GnRH+cells was significantly increased in ARC(P<0.05)but was decreased in PVN and ME(P<0.05).The number of Kisspeptin+cells in ARC and PeN was significantly decreased(P<0.05),and the levels of E 2 and P 4 were significantly decreased(P<0.05).【Conclusion】NKB promoted puberty initiation by modulating the expressions of Kisspeptin and GnRH in hypothalamus and increasing the concentrations of E 2 and P 4 in serum.
作者
司文宇
宋爽
康铁柱
姚郅璆
叶菁
于童
方富贵
SI Wenyu;SONG Shuang;KANG Tiezhu;YAO Zhiqiu;YE Jing;YU Tong;FANG Fugui(Anhui Provincial Laboratory of Animal Genetic Resources Protection and Breeding,College of Animal Sciences and Technology,Anhui Agricultural University,Hefei,Anhui 230036,China)
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2020年第4期10-17,26,共9页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家自然科学基金项目(31472096)。
