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

Abstract

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

CLC Number:

S831.3

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.

Figures/Tables 10

Table 1

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

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