兔原始生殖细胞体外培养的研究

doi:10.11843/j.issn.0366-6964.2020.10.012

摘要/Abstract

摘要： 旨在探索兔原始生殖细胞（primordial germ cells，PGCs）体外分离培养的最佳条件，从而建立成熟稳定的兔PGCs体外分离培养方法。本研究首先通过两种不同的体外分离法（胰酶消化法、机械法）和3种不同的传代法（胰酶消化法、机械法、连同饲养层消化法）探索第14~18天胎兔的PGCs体外分离传代的最佳方式，另将培养液分为A、B、C、D 4组，以D组为对照组，探究不同细胞因子浓度对兔PGCs形态变化及集落形成的影响。其次，利用碱性磷酸酶染色（alkaline phosphatase staining，AKP）法对兔PGCs进行鉴定染色；实时荧光定量（real-time polymerase chain reaction，RT-PCR）检测转录因子Oct-4的表达。结果表明，机械法分离得到的兔PGCs集落数量是酶消化法分离的2.2倍，而兔PGCs经不同的消化法传代发现，酶消化法、连同饲养层消化法、机械法在兔胚胎成纤维细胞（rabbit embryo fibroblast，REF）饲养层上分别成功传至P2、P2、P4代。B组PGCs培养液（基础液+10%胎牛血清（fetal bovine serum，FBS）+2 ng·mL^-1转化生长因子-β1（transforming growth factor-β1，TGF-β1）+4 ng·mL^-1碱性成纤维细胞生长因子（basic fibroblast growth factor， bFGF）+10 ng·mL^-1白血病抑制因子（leukemia inhibitory factor，LIF））所获得的集落数量最多，具有较好的集落形态，保持未分化的时间较长。AKP染色结果显示，PGCs集落呈红黑色； RT-PCR结果显示，体外分离培养的兔PGCs表达转录因子Oct-4。结果显示，兔原代PGCs最适采用机械分离法和机械传代法进行体外分离传代，适宜浓度的细胞因子添加至兔PGCs培养液中有利于兔PGCs在体外保持较多的集落数量和较长时间的未分化状态。本研究通过筛选和优化兔PGCs体外培养方法，为进一步建立稳定成熟兔PGCs细胞系奠定技术基础。

关键词: 兔, 原始生殖细胞, 分离方法, 传代方法, 细胞因子

Abstract: The purpose of the present study was to explore the optimal condition for the isolation and culture of rabbit primordial germ cells(PGCs) in vitro, and to establish a mature and stable method for the isolation and culture of rabbit PGCs in vitro. Firstly, trypsin digestion and mechanical methods were used to explore the best method for isolating PGCs from 14- to 18-day-old fetal rabbits in vitro, and trypsin digestion, mechanical method and digestion with the feeder layer were used to explore the optimal way to passage the isolated PGCs. Then, 4 different culture media(A, B, C and D (control)) were used to explore the effect of different concentrations of cytokines on the morphological changes and colony formation of rabbit PGCs. Secondly, rabbit PGCs were identified by alkaline phosphatase staining (AKP), and the expression of transcription factor Oct-4 was detected by real-time PCR (RT-PCR). The results showed that the number of rabbit PGCs colonies isolated by mechanical method was 2.2 times higher than those isolated by trypsin digestion method. Rabbit embryo fibroblasts (REF) were passaged to the second generation (P2) using trypsin digestion method and digestion with the feeder layer method, and REF were passaged to the fourth generation (P4) using the mechanical method. The largest number of colonies and better colony morphology were observed when PGCs were cultured in medium B(basic medium＋10% fetal bovine serum (FBS) ＋2 ng·mL^-1 of transforming growth factor-β1 (TGF-β1) ＋4 ng·mL^-1of basic fibroblast growth factor (bFGF) ＋10 ng·mL^-1leukemia inhibitory factor (LIF)). Rabbit PGCs remained undifferentiated for a longer time when cultured in medium B. AKP staining showed that the isolated PGCs was positive in red and black, and expression of Oct-4 gene was also detected in the isolated PGCs by RT-PCR. The results showed that mechanical isolation method and mechanical passage method were the optimal ways to isolate and passage rabbit primary PGCs, respectively. Supplementation with appropriate concentration of cytokines into culture medium was beneficial for rabbit PGCs to maintain a large number of colonies and undifferentiated state for a longer time in vitro. In the present study, the culture methods of rabbit PGCs in vitro were screened and optimized, which would lay a technical foundation for the further establishment of a stable and mature rabbit PGCs cell line.

Key words: Oryctolagus cuniculus, primordial germ cells (PGCs), isolation methods, passage methods, cytokines

中图分类号:

S829.1

吕海淼, 朱邯豫, 彭展, 闫琛博, 杨德新, 胡文举, 丁雪粉, 王新庄. 兔原始生殖细胞体外培养的研究[J]. 畜牧兽医学报, 2020, 51(10): 2443-2452.

Lü Haimiao, ZHU Hanyu, PENG Zhan, YAN Chenbo, YANG Dexin, HU Wenju, DING Xuefen, WANG Xinzhuang. Study on the Culture of Rabbit (Oryctolagus cuniculus) Primordial Germ Cells in Vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10): 2443-2452.

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