沉默Ttf1对神经元细胞及雌性大鼠弓状核内Kiss1和GnRH基因表达的影响

doi:10.3969/j.issn.1674-8115.2020.11.002

上海交通大学学报(医学版) ›› 2020, Vol. 40 ›› Issue (11): 1447-1453.doi: 10.3969/j.issn.1674-8115.2020.11.002

沉默Ttf1对神经元细胞及雌性大鼠弓状核内Kiss1和GnRH基因表达的影响

臧少莲，尹小琴，吕拥芬，许丽雅，郭盛，李嫔

上海交通大学附属儿童医院，上海市儿童医院内分泌科，上海200333

出版日期:2020-11-28 发布日期:2021-01-13
通讯作者: 李嫔，电子信箱：lipin21@126.com。
作者简介:臧少莲（1994—），女，硕士生；电子信箱：shaolianz@163.com。
基金资助:
国家自然科学基金（81871131）；上海交通大学医工交叉基金（YG2015MS39）；金磊儿科内分泌中青年医师成长科研基金（PEGRF201506002）；上海市儿童医院院级面上项目（2016YMS003）；上海市卫生和计划生育委员会面上项目（201640140）。

Effects of silenced Ttf1 on genes expression of Kiss1 and GnRH in neuronal cells and arcuate nucleus of female rats

ZANG Shao-lian, YIN Xiao-qin, LÜ Yong-fen, XU Li-ya, GUO Sheng, LI Pin

Department of Endocrinology, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200333, China

Online:2020-11-28 Published:2021-01-13
Supported by:
National Natural Science Foundation of China (81871131); Medical Engineering Cross Research Fund of Shanghai Jiao Tong University (YG2015MS39); Jin Lei Pediatric Endocrinology Growth Research Fund for Young Physicians (PEGRF201506002); General Project of Shanghai Children's Hospital (2016YMS003); General Program of Shanghai Municipal Commission of Health and Family Planning (201640140).

摘要/Abstract

摘要： 目的·探讨抑制神经元细胞和雌性大鼠下丘脑弓状核中甲状腺转录因子1（thyroid transcription factor 1，Ttf1）基因的表达对亲吻素（Kiss1）和促性腺激素释放激素（gonadotropin-releasing hormone，GnRH）基因表达的影响。方法·针对大鼠Ttf1基因的mRNA序列分别设计3对特异性的干扰序列（TTF1-shRNA1、TTF1-shRNA2、TTF1-shRNA3），包装成慢病毒质粒。以神经元细胞ND7-23为体外模型，采用real-time PCR及Western blotting技术筛选干扰效率最高的慢病毒质粒（LV-TTF1-shRNA），转染72 h后检测Kiss1和GnRH基因的表达。将LV-TTF1-shRNA和空载质粒注射到21日龄雌性大鼠的下丘脑弓状核内，分别在幼年期（25日龄）、青春发育早期（35日龄）、成年期（42日龄）检测弓状核内Kiss1和GnRH基因的表达变化并观察雌鼠的阴门开放时间。结果·慢病毒质粒转染ND7-23细胞72 h后，LV-TTF1-shRNA2组、LV-TTF1-shRNA3组的Ttf1 mRNA及蛋白表达均显著减少（均P<0.05），其中LV-TTF1-shRNA3干扰效果最佳。采用LV-TTF1-shRNA3抑制ND7-23细胞中Ttf1表达后，Kiss1和GnRH基因的mRNA表达均显著降低（均P<0.05）。雌鼠弓状核内双侧显微注射LV-TTF1-shRNA3后，25日龄、35日龄、42日龄大鼠弓状核Kiss1和GnRH的mRNA均显著降低（均P<0.05），35日龄和42日龄时KISS1蛋白水平明显降低，且雌鼠阴门开放时间延迟。结论·在细胞水平和雌性大鼠弓状核内抑制Ttf1基因表达均使得Kiss1和GnRH基因表达显著下调，也导致雌鼠阴门开放时间明显延迟。

关键词: 甲状腺转录因子1, 亲吻素基因, 促性腺激素释放激素, RNA干扰, 慢病毒载体, 青春期发育

Abstract:

Objective · To explore the effects of inhibiting thyroid transcription factor 1 (Ttf1) expression on Kiss1 and gonadotropin-releasing hormone (GnRH) expressions in neuronal cells and hypothalamic arcuate nucleus (ARC) of female rats. Methods · Three shRNAs (TTF1-shRNA1, TTF1-shRNA2, and TTF1-shRNA3) interference sequences were designed for rat Ttf1 gene and were constructed into lentiviral vectors. The efficiency of LV-TTF1-shRNA was detected by real-time PCR and Western blotting in neuronal cell line ND7-23. And the expressions of Kiss1 and GnRH were tested 72 h after transfection. Moreover, LV-TTF1-shRNA or the blank lentiviral vector was injected into 21-day female rats’ ARC. Kiss1 and GnRH expressions were detected at the juvenile (25-day), early puberty (35-day) and adult (42-day) stages. Meanwhile, vaginal opening time of the rats was recorded. Results · Seventy-two hours after lentivirus transfection in ND7-23 cells, the mRNA and protein expressions of Ttf1 decreased significantly in LV-TTF1-shRNA2 group and LV-TTF1-shRNA3 group (all P<0.05), particularly in LV-TTF1-shRNA3 group. The expressions of Kiss1 and GnRH mRNA reduced significantly after LV-TTF1-shRNA3 being transfected in ND7-23 cells (both P<0.05). After injection of LV-TTF1-shRNA3 in female rats’ ARC, mRNA abundance of Kiss1 and GnRH showed evident reduction at 25-day, 35-day and 42-day stages (all P<0.05). And KISS1 protein reduced significantly at 35-day and 42-day stages. Female rats transfected with LV-TTF1-shRNA3 showed delayed vaginal opening. Conclusion · Inhibition of Ttf1 gene expression not only significantly reduces the expression of Kiss1 and GnRH in neuronal cells and female rats’ ARC, but also causes female rats vaginal opening delay.

Key words: thyroid transcription factor 1 (TTF1), Kiss1 gene, gonadotropin-releasing hormone (GnRH), RNA interference (RNAi), lentiviral vector, puberty

中图分类号:

R725.8

臧少莲，尹小琴，吕拥芬，许丽雅，郭盛，李嫔. 沉默Ttf1对神经元细胞及雌性大鼠弓状核内Kiss1和GnRH基因表达的影响[J]. 上海交通大学学报(医学版), 2020, 40(11): 1447-1453.

ZANG Shao-lian, YIN Xiao-qin, LÜ Yong-fen, XU Li-ya, GUO Sheng, LI Pin. Effects of silenced Ttf1 on genes expression of Kiss1 and GnRH in neuronal cells and arcuate nucleus of female rats[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2020, 40(11): 1447-1453.

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沉默Ttf1对神经元细胞及雌性大鼠弓状核内Kiss1和GnRH基因表达的影响

Effects of silenced Ttf1 on genes expression of Kiss1 and GnRH in neuronal cells and arcuate nucleus of female rats

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