猪精原干细胞体外分离培养及诱导分化的研究

doi:10.11843/j.issn.0366-6964.2014.07.011

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (7): 1104-1112.doi: 10.11843/j.issn.0366-6964.2014.07.011

猪精原干细胞体外分离培养及诱导分化的研究

陈庭锋¹,王霄燕¹,李东¹,施青青¹，张蕾¹,邱峰龙¹,刘志永¹,庄勋²,卞桂华²，宋成义¹,李碧春^1*

（1.扬州大学动物科学与技术学院，扬州 225009； 2.江苏农牧科技职业学院，泰州 225300）

收稿日期:2013-12-27 出版日期:2014-07-23 发布日期:2014-07-23
通讯作者: 李碧春，教授，E-mail：yubcli@yzu.edu.cn
作者简介:陈庭锋(1987-)，男，河南固始人，硕士，主要从事精原干细胞培养方面研究，E-mail：ctf20070702664@126.com
基金资助:
转基因生物新品种培育重大专项（2011ZX08006-005）

Study on the Culture and Induced Differentiation of Spermatogonial Stem Cells in Porcine

CHEN Ting-feng¹，WANG Xiao-yan¹，LI Dong¹，SHI Qing-qing¹，ZHANG Lei¹，QIU Feng-long¹，LIU Zhi-yong¹，ZHUANG Xun²，BIAN Gui-hua¹，SONG Cheng-yi¹，LI Bi-chun^1*

(1.College of Animal Science and Technology，Yangzhou University，Yangzhou 225009， China；2.Jiangsu Agri-animal Husbandry Vocational College，Taizhou 225300， China)

Received:2013-12-27 Online:2014-07-23 Published:2014-07-23

摘要/Abstract

摘要：

本研究旨在建立猪精原干细胞（Porcine spermatogonial stem cells，pSSCs）体外分离和诱导分化，并探索pSSCs定向分化及分化过程中关键基因的表达情况。采用差速贴壁法分离pSSCs和支持细胞（Sertoli），采用生化和免疫学方法鉴定其干细胞特性；传至3代后通过添加不同诱导剂，诱导SSCs向脂肪细胞、神经元样细胞和成骨细胞分化，并通过生化染色、qRT-PCR和免疫细胞化学检测诱导效果。结果表明：分离得到的pSSCs在饲养层上生长良好，碱性磷酸酶（AKP）及SOX2、integrinα6、SSEA1、Dazl抗体鉴定均呈阳性，表明SSCs处在未分化状态。诱导8 d后，甲苯胺蓝染色及NSE抗体鉴定成阳性，成功诱导SSCs为神经元样细胞；诱导16 d 后，ALP、Von Kossa染色及Collagon l抗体鉴定均呈阳性，诱导SSCs为成骨细胞；诱导22 d 后，油红O染色可见脂滴被染成红色，诱导SSCs成为脂肪细胞。在SSCs诱导过程中，qRT-PCR结果显示，Nestin和β-tubulin在6 d左右表达量最高，Cbfα1和Osterix在12 d左右表达量最高，PPARγ和C/EBPα在18 d左右表达量最高。本研究成功分离获得pSSCs，并且pSSCs可分别定向诱导分化为成骨细胞、神经元样细胞和脂肪细胞，表达细胞特有标记基因。

Abstract:

The aim of this study was to establish the culture system of pSSC in vitro，and explore the expression of these key genes during the induced differentiation.Using the methods of differential adherent，we had isolated pSSCs from the supporting(Sertoli) cells.In addition，we also had identified the stem characteristics by the biochemical and immunological methods.When it grew to generation 3，the SSCs had been induced into the differentiation of adipocytes，neuron-like cells and osteoblast，through the addition of different chemical reagents；moreover，the results of induction also had been detected by the biochemical staining，quantitative PCR(qPCR) and immunocytochemistry.The results showed that：the isolated pSSCs grew well in the feeder layer，and the identification of alkaline phosphatase(AKP) and SOX2，integrinα6，SSEA1，Dazl antibodies were positive，suggesting the pSSCs were in the undifferentiated state.After 8 days of induction，the identification of toluidine blue staining and NSE antibodies were positive，suggesting that the pSSCs had been induced into the neuron-like cells successfully.After 16 days of induction，the identification of ALP，Von Kossa staining and Collagon l antibodies were positive，indicating it had been induced into the osteoblasts.Twenty-two days later，the lipid droplets had been stained into red by oil red O，demonstrated that the pSSCs had been induced into adipocytes.During the process of the pSSCs induction，the results of qPCR demonstrated that，the expression of Nestin and β-tubulin were highest at day 6，the expression of Cbfα1 and Osterix were highest at day 12，however，the mRNA expression of PPARγ and C/EBPα were highest at day 18.In this study，we had obtained the pSSCs successfully，and the pSSCs also had been induced into the neuron-like cells，osteoblasts and adipocytes，separately，comparing the expression of cell-special marker genes.

中图分类号:

S828
S814

陈庭锋,王霄燕,李东,施青青，张蕾,邱峰龙,刘志永,庄勋,卞桂华，宋成义,李碧春. 猪精原干细胞体外分离培养及诱导分化的研究[J]. 畜牧兽医学报, 2014, 45(7): 1104-1112.

CHEN Ting-feng，WANG Xiao-yan，LI Dong，SHI Qing-qing，ZHANG Lei，QIU Feng-long，LIU Zhi-yong，ZHUANG Xun，BIAN Gui-hua，SONG Cheng-yi，LI Bi-chun. Study on the Culture and Induced Differentiation of Spermatogonial Stem Cells in Porcine[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(7): 1104-1112.

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猪精原干细胞体外分离培养及诱导分化的研究

Study on the Culture and Induced Differentiation of Spermatogonial Stem Cells in Porcine

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