Hsd3β2基因RNA干扰载体构建及对鸡ESCs向PGCs分化的调控

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摘要摘要原始生殖细胞(primordial germ cells, PGCs)作为配子的原始祖细胞，参与胚胎生殖细胞的发育过程。类固醇激素通路中3β羟基类固醇脱氢酶2 (3β-hydroxysteroid dehydrogenase 2, Hsd3β2)能促进细胞分化，但其对原始生殖细胞的形成具体调控机制还不得而知。本研究旨在通过构建类固醇激素合成过程中关键基因Hsd3β2的短发夹RNA(short hairpin RNA， shRNA)干扰载体并研究其对鸡(Gallus gallus)胚胎干细胞分化为原始生殖细胞的影响。通过慢病毒包装Hsd3β2感染鸡胚胎干细胞并进行RNA干扰(RNA interference, RNAi)诱导，分别于2、4、6 d观察细胞形态变化并收集细胞样品，qRT-PCR检测Hsd3β2、生殖标记基因鸡Vasa基因同系物(chicken vasa homologue, Cvh)、鸡KIT原癌基因受体酪氨酸激酶(chicken KIT proto-oncogene receptor tyrosine kinase, C-kit)及甾类激素合成信号通路下游基因细胞色素P450亚酶(cytochrome P450 subenzyme, Cyp1a1)、葡萄糖醛酸转移酶1A1 (UDP glucuronosyltransferase family 1 member A1, Ugt1a1)和17β羟基类固醇脱氢酶7(17beta-hydroxysteroid dehydrogenase7, Hsd17b7)表达；流式细胞检测诱导产生类胚体阳性率；在鸡胚孵化过程中鸡胚注射慢病毒干扰载体，孵化4.5 d后收集生殖嵴，qRT-PCR检测Cvh、C-kit及下游基因Cyp1a1、Ugt1a1和Hsd17b7表达；流式细胞分析原始生殖细胞生成效率。本实验成功构建sh-Hsd3β2慢病毒载体，挽救实验表明过表达Hsd3β2能使shRNA引起的基因表达下降回升；在体外诱导过程和鸡胚孵化过程中，qRT-PCR结果表明干扰Hsd3β2后，甾类激素合成信号通路中的下游基因的表达显著下调(P＜0.01)；在RA诱导实验过程中，随诱导时间推进，类胚体数量逐渐增加。Hsd3β2抑制后，类胚体形成数量减少。Cvh、C-kit在诱导过程中上调表达，但Hsd3β2干扰后，其表达显著降低(P＜0.01)，流式细胞分析表明干扰Hsd3β2后，诱导4d后产生类胚体阳性细胞(3.27±0.19)%显著低于对照组(7.39±0.09)%；体内实验结果表明干扰Hsd3β2后，Cvh、C-kit表达显著降低(P＜0.01)，生殖细胞数量显著减少。本研究结果表明Hsd3β2能通过甾类激素合成信号通路促进原始生殖细胞的形成，为研究该通路对原始生殖细胞形成具体机制提供理论基础。

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	张晨
	王曼
	金晶
	何娜娜
	李婷婷
	孙新宇
	胡菜
	周舒简
	孙长花
	左其生
	张亚妮
	李碧春

关键词 ：鸡, 胚胎干细胞, 3β羟基类固醇脱氢酶2基因(Hsd3β2), 甾类激素合成信号通路, 分化

Abstract：Abstract Primordial germ cells (PGCs) are the progenitor cells of gametes involving in the development of embryonic germ cells (ESCs). 3β-hydroxysteroid dehydrogenase 2 (Hsd3β2) can promote cell differentiation in the steroid hormone pathway, but its specific regulation mechanism on the formation of PGCs is still unknown. The study aims to construct a short hairpin RNA (shRNA) interfering vector of Hsd3β2, a key gene in the process of steroid hormone synthesis and to observe its effect on the differentiation of chicken (Gallus gallus) ESCs into PGCs. Hsd3β2 was sythesized, amplified, and ligated into the lentiviral plasmid pGMLV-SC5. Gene sequencing was done to identify the successful construction of the vector. DF1 was transfected by 3 vectors and a control vector to choose the best one for the next lentiviral package. On the basis of retinoic acid (RA) induction, ESCs were transfected by sh-Hsd3β2 and the cell morphology was observed at 2, 4 and 6 d, respectively. Cell samples were collected for further experiments. qRT-PCR was used to detect the expression of Hsd3β2, marker genes chicken KIT proto-oncogene receptor tyrosine kinase (Cvh) and chicken KIT proto-oncogene receptor tyrosine kinase (C-kit), downstream genes cytochrome P450 subenzyme (Cyp1a1), UDP glucuronosyltransferase family 1 member A1 (Ugt1a1) and 17beta-hydroxysteroid dehydrogenase7 (Hsd17b7). Flow cytometry in vitro showed the number of the positive cells. After injecting the vector in vivo, genital ridges were collected in 4.5 d. qRT-PCR was used to detect the expression of Hsd3β2, marker genes and downstream genes. Flow cytometry was used to analyze the number of PGCs. PGCs were observed by optical microscopy and periodic acid-schiff stain (PAS). The target gene was successfully ligated into the lentiviral vector. The vector had a high efficiency and shRNA-2 has a better interference efficiency in DF1. qRT-PCR showed that after transfecting sh-Hsd3β2 and pc-Hsd3β2, the expression of Hsd3β2 increased. After packaging with lentiviral, the viral titer was 5×106 TU/ mL. qRT-PCR showed that after interfering Hsd3β2 in vitro/in vivo, the expression of downstream genes Cyp1a1, Ugt1a1 and Hsd17b7 in the steroid hormone signaling pathway was significantly decreased (P＜0.01). After inhibiting Hsd3β2 during the induction experiment, the number of embryoid body decreased and embryoid body appeared later. qRT-PCR showed that marker genes Cvh, C-kit significantly decreased (P＜0.01). Flow cytometry indicated that after inhibiting Hsd3β2, the number of positive cells (3.27±0.19)% compared with control group (7.39±0.09)% significantly decreased in vitro. In vivo, after inhibiting Hsd3β2, PAS staining showed that the number of PGCs had been descreased. qRT-PCR indicated that marker genes decreased (P＜0.01). Flow cytometry showed that after inhibiting the expression of Hsd3β2, DDX4+ positive cells decreased.Hsd3β2 could regulate the steroid hormone synthesis pathway to positively affect the differentiation of ESCs to PGCs.

Key words： Chicken Embryonic stem cells (ESCs) 3β-hydroxysteroid dehydrogenase 2 gene (Hsd3β2) Steroid hormone pathway Differentiation

收稿日期: 2018-01-17 出版日期: 2018-08-06

ZTFLH:

S814.6

基金资助:;国际合作重点研发专项;扬州大学“高端人才支持计划”项目;江苏省畜牧学优势学科建设工程项目;扬州大学2017年大学生学术科技创新基金项目

通讯作者: 李碧春 E-mail: yubcli@yzu.edu.cn

引用本文:

张晨王曼金晶何娜娜李婷婷孙新宇胡菜周舒简孙长花左其生张亚妮李碧春. Hsd3β2基因RNA干扰载体构建及对鸡ESCs向PGCs分化的调控[J]. , 2018, 26(9): 1567-1576.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2018/V26/I9/1567

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