洛岛红鸡卵黄囊源性间充质干细胞的分离培养及其分化潜能研究

doi:10.11843/j.issn.0366-6964.2025.03.025

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (3): 1252-1263.doi: 10.11843/j.issn.0366-6964.2025.03.025

洛岛红鸡卵黄囊源性间充质干细胞的分离培养及其分化潜能研究

闫炎¹^,²(), 刘晏辰²(), 王仲发², 李旻娟², 何玉楠², 关伟军²^,*(), 姜运良¹^,*()

1. 山东农业大学动物科技学院, 泰安 271000
2. 中国农业科学院北京畜牧兽医研究所, 北京 100193

收稿日期:2024-06-26 出版日期:2025-03-23 发布日期:2025-04-02
通讯作者: 关伟军,姜运良 E-mail:17853651215@163.com;401979899@qq.com;guanweijun@caas.cn;zhaojy@sdau.edu.cn
作者简介:闫炎(1998-)，女，山东济宁人，硕士生，主要从事间充质干细胞与动物生物技术研究，E-mail：17853651215@163.com
刘晏辰(1997-)，男，黑龙江佳木斯人，博士生，主要从事畜牧相关研究，E-mail: 401979899@qq.com
第一联系人：
闫炎和刘晏辰为同等贡献作者
基金资助:
国家重点研发计划(2021YFD1300100；2021YFD1200303)

Isolation, Culture and Differentiation Potential of Mesenchymal Stem Cells of Yolk Sacs from Rhode Island Red Chicken

YAN Yan¹^,²(), LIU Yanchen²(), WANG Zhongfa², LI Minjuan², HE Yunan², GUAN Weijun²^,*(), JIANG Yunliang¹^,*()

1. College of Animal Science and Technology, Shandong Agricultural University, Taian 271000, China
2. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received:2024-06-26 Online:2025-03-23 Published:2025-04-02
Contact: GUAN Weijun, JIANG Yunliang E-mail:17853651215@163.com;401979899@qq.com;guanweijun@caas.cn;zhaojy@sdau.edu.cn

摘要/Abstract

摘要：

旨在体外获得并培养洛岛红鸡卵黄囊间充质干细胞(chicken yolk sac mesenchymal stem cell，cYS-MSCs)，检测其体外增殖能力和生物学特性。本研究选取已孵化12天的健康洛岛红鸡受精蛋的卵黄囊进行组织贴壁培养，选择第5代cYS-MSCs进行免疫荧光和RT-PCR检测表面标记基因；选择第3代、第9代和第15代cYS-MSCs，每代次设置3个重复，绘制生长曲线、计算群体倍增时间和克隆形成率；通过核型分析检测遗传稳定性；选择第9代cYS-MSCs进行成脂、成软骨和成骨诱导，通过特异性染色和RT-PCR检测多向分化能力。结果，分离培养的细胞为长梭形，呈漩涡状或平行式生长；免疫荧光结果显示CD29、CD73、CD90和CD166呈阳性表达，而造血干细胞标记基因CD34和泛白细胞标记基因CD45不表达；RT-PCR结果显示CD29、CD44、CD73、CD90和CD166表达，而CD34和CD45不表达；cYS-MSCs呈“S”型增长，且第3代的群体倍增时间极显著低于第9代(P＜0.001)，第9代群体倍增时间极显著低于第15代(P＜0.001)，第3代的克隆形成率极显著高于第9代(P＜0.001)，第9代极显著高于第15代(P＜0.001)；染色体核型分析结果显示经过连续传代的cYS-MSCs染色体数量、形态正常(2n=78，zz)；诱导分化的cYS-MSCs，特异性染色和RT-PCR结果显示cYS-MSCs可被成功诱导分化为成脂肪细胞、成软骨细胞和成骨细胞。综上，本研究成功从鸡胚卵黄囊中分离获得MSCs，且能在体外培养增殖，并且分离的cYS-MSCs具有MSCs的生物学特性和多向分化潜能，其多向分化潜能预示细胞移植的前景，可为洛岛红鸡种质资源保存提供依据和潜在可利用资源。

关键词: 卵黄囊, 间充质干细胞, 洛岛红鸡, 生物学特性, 分化潜能

Abstract:

The study aimed to obtain and cultivate Rhode Island Red yolk sac mesenchymal stem cells (cYS-MSCs), then evaluate proliferative capacity and biological properties in vitro. The yolk sac of fertilized Rhode Island Red eggs, which had been incubated for 12 days, was selected for tissue adherent culture. The 5th generation of cYS-MSCs was chosen for immunofluorescence and RT-PCR analyses to detect surface marker genes. Additionally, the 3rd, 9th, and 15th generations of cYS-MSCs were selected, with 3 replicates established for each generation, to plot growth curves, calculate population doubling time, and assess colony formation rates. Genetic stability was evaluated through karyotyping. The 9th generation of cYS-MSCs was further selected for adipogenic and cartilage induction, with multidirectional differentiation capabilities assessed using specific staining and RT-PCR. The isolated cells were characterized by a long, spindle-shaped morphology, growing in either a swirling or parallel pattern. Immunofluorescence results indicated the expression of CD29, CD73, CD90, and CD166, while the hematopoietic stem cell marker gene CD34 and the panleukocyte marker gene CD45 were not expressed. Additionally, RT-PCR results confirmed the expression of CD29, CD44, CD73, CD90, and CD166, with CD34 and CD45 remaining unexpressed. The cYS-MSCs exhibited an ′S′ type growth pattern, with the population doubling time of the 3rd generation being extremely significantly lower than that of the 9th generation (P < 0.001). Furthermore, the population doubling time of the 9th generation was extremely significantly lower than the 15th generation (P < 0.001). The cloning rate of the 3rd generation was extremely significantly higher than the 9th generation (P < 0.001), and the cloning rate of 9th generation was also extremely significantly higher than the 15th generation (P < 0.001). Karyotype analysis revealed that the number and morphology of cYS-MSCs were normal (2n=78, zz). Specific staining and RT-PCR results demonstrated that cYS-MSCs could be successfully induced to differentiate into adipoblasts, chondroblasts, and osteoblasts. In conclusion, MSCs were successfully isolated from the yolk sac of chicken embryos, demonstrating the ability to be cultured and proliferated in vitro. The isolated cYS-MSCs exhibited the biological characteristics and multidirectional differentiation potential typical of MSCs. This differentiation potential suggests promising prospects for cell transplantation, providing a foundation and potential resources for the conservation of Rhode Island Red germplasm resources.

Key words: yolk sacs, mesenchymal stem cells, Rhode Island Red chicken, biological characteristics, differentiation potential

中图分类号:

S831.3

闫炎, 刘晏辰, 王仲发, 李旻娟, 何玉楠, 关伟军, 姜运良. 洛岛红鸡卵黄囊源性间充质干细胞的分离培养及其分化潜能研究[J]. 畜牧兽医学报, 2025, 56(3): 1252-1263.

YAN Yan, LIU Yanchen, WANG Zhongfa, LI Minjuan, HE Yunan, GUAN Weijun, JIANG Yunliang. Isolation, Culture and Differentiation Potential of Mesenchymal Stem Cells of Yolk Sacs from Rhode Island Red Chicken[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1252-1263.

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基因 Gene	引物序列(5'→3') Primer sequence	产物长度/bp Size	退火温度/℃ Tm
GAPDH	F: AGGTGCTGAGTATGTTGTG R: CGCTGGGATGATGTTCTG	358	56.8
CD29	F: TGAATGACACGCAGGAAGATGGAAG R: AGGCAGGGCTGTTAGGTTCTCC	219	59.3
CD44	F: CCTCCTGGCACTGGCATTGATC R: CACTCCACTCTTCATGTCACCATCC	271	58.9
CD73	F: GGCATCGTTGGCTACACTACACAG R: GCTTGTACCACTGGCACCTTCC	326	54.2
CD90	F: ACCAAGGACAACAGGAAGCACATC R: GGTGTTCTGGATCAAGAGGCTGAAG	258	57.9
CD166	F: ACCAGCAGTGCAATGGACAGTTAC R: GCAACCAGCAGAAGACCGACTAC	294	58.5
CD34	F: GCCACTCGCATCCAGAGAACAC R: GTCAGCATCCCCTTCGCATATCG	326	61.5
CD45	F: GTCATGGTTACTCGCTGTGAGGAG R: CGTCGGAGTTTGAGAAGGAGATGTG	263	58.7
LPL	F: GTGACCAAGGTAGACCAGCCATTC R: TCGCCTGACTTCACTCTGACTCTC	248	54.3
PPAR-γ	F: CGAATGCCACAAGCGGAGAAGG R: CACTGCCTCCACAGAGCGAAAC	330	59.4
ACAN	F: CCTCAGAGACCTACGATGT R: TGTGGTGCTGGTAGATGG	258	57.8
SOX9	F: GGAGGCTGCTGAATGAGA R: CTGCTGATGCCGTAGGTA	550	60.4
OPN	F: GGTAAGGATGGTCGCAATGGTCTC R: GGGTCAGCAGGGTCTCAATTTGG	256	59.1
COLⅠ	F: AGCATCAGTGGAGGTAGTT R: CAGGCATTCCAGGCATTC	355	58.1

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洛岛红鸡卵黄囊源性间充质干细胞的分离培养及其分化潜能研究

Isolation, Culture and Differentiation Potential of Mesenchymal Stem Cells of Yolk Sacs from Rhode Island Red Chicken

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