猪类胚胎生殖细胞体外诱导分化为神经细胞的研究

doi:10.3864/j.issn.0578-1752.2013.06.022

摘要/Abstract

摘要： 【目的】探讨猪类胚胎生殖细胞向神经细胞分化的潜能。【方法】采用性腺-中肾区基质细胞与猪原始生殖细胞共培养的方法得到猪类胚胎生殖细胞，通过直接分化的方法进行神经细胞定向诱导。【结果】（1）与小鼠成纤维细胞作为饲养层相比，性腺-中肾区基质细胞作为饲养层同样能够支持猪类胚胎生殖细胞生长，二者对猪类胚胎生殖细胞的增殖能力不存在显著影响；（2）猪类胚胎生殖细胞有较强的碱性磷酸酶活性，Q-PCR结果显示多能性基因Oct4、Sox2及Nanog表达，增殖能力呈现“S”型，即生长的潜伏期、对数期及平台期，猪类胚胎生殖细胞经体外悬浮培养能够形成拟胚体；（3）经过诱导猪类胚胎生殖细胞能够分化形成表达神经干细胞、神经元及神经胶质细胞标记物的多种神经细胞类型。【结论】从早期性腺中成功分离培养得到了猪类胚胎生殖细胞，通过简单、快速的神经细胞分化的诱导体系证实了猪类胚胎生殖细胞具有体外定向分化为多种神经谱系细胞的能力。

关键词: 类胚胎生殖细胞 , 体外诱导 , 神经细胞 , 猪

Abstract: 【Objective】The objective of the study is to detect the potential of porcine putative embryonic germ cells differentiating into nerve cells. 【Method】Co-culture of Gonad-mesonephros (GM) region stromal cells and primordial germ cells was used to get porcine putative embryonic germ cells. Directional differentiation method was adopted for neural differentiation. 【Result】 Compared with MEF , GM region stromal cells as feeder could support the growth of porcine putative embryonic germ cells with no significant difference in the proliferation of embryonic germ cells. Porcine putative embryonic germ cells had strong AP activity, Q-PCR results indicated the expression of Oct4, Sox2 and Nanog genes. Proliferating ability of porcine putative embryonic germ cells showed a "S" type, namely the growth of latency, logarithmic phase and plateau phase. Embryoid body could be derived after suspending culture of porcine putative embryonic germ cells in vitro. After induction, porcine putative embryonic germ cells could differentiate into a variety of neural cell types with expression of neural stem cells, neurons and glial cells markers.【Conclusion】These results indicate that porcine putative embryonic germ cells can be derived from porcine early gonadal and have the ability of differentiating into a variety of neural lineage cells in vitro.

Key words: putative embryonic germ cells , induce in vitro , nerve cells , pig

丛义梅, 马婧, 孙瑞珍, 王健宇, 薛冰华, 胡魁, 尹智, 刘忠华. 猪类胚胎生殖细胞体外诱导分化为神经细胞的研究[J]. 中国农业科学, 2013, 46(6): 1272-1279.

CONG Yi-Mei, MA Jing, SUN Rui-Zhen, WANG Jian-Yu, XUE Bing-Hua, HU Kui, YIN Zhi, LIU Zhong-Hua. Study on Induction of Porcine Putative Embryonic Germ Cells into Nerve Cells in vitro[J]. Scientia Agricultura Sinica, 2013, 46(6): 1272-1279.

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参考文献

[1]Evans M J, Kaufman M H, Establishment in culture of pluripotential cells from mouse embryos. Nature, 1981, 292(5819): 154-156.

[2]Martin G R, Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proceeding of National Academy Science of United State of America, 1981, 78(12), 7634-7638.

[3]Matsui Y, Zsebo K, Hogan B, Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell, 1992, 70(5): 841-847.

[4]Resnick J, Bixler L, Cheng L, Donovan P, Long-term proliferation of mouse primordial germ cells in culture. Nature, 1992, 359 (6395): 550-551.

[5]Takahashi K, Yamanaka S,. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126: 663-676.

[6]Evans M J, Notatianni E, Laurie S, Moor R M. Derivation and preliminary characterization of pluripotent cell lines from porcine and bovine blastocyst. Theriogenology, 1990, 33(1): 125-128.

[7]Kim H S, Son H Y, Kim S, Lee G S, Park C H, Kang S K. Isolation and initial culture of porcine inner cell masses derived from in vitro-produced blastocysts. Zygote, 2007, 15(1): 55-63.

[8]赵颖, 孔庆然, 刘忠华. 猪胚胎干细胞研究进展. 中国细胞生物学学报, 2010, 32(5): 815-821.

Zhao Y, Kong Q R, Liu H Z. Current progress in porcine embryonic stem cells. Chinese Journal of Cell Biology, 2010, 32(5): 815-821. (in Chinese)

[9]Chen L R, Shiue Y L, Bertolini L. Establishment of pluripotent cell lines from porcine preimplantation embryos. Theriogenology, 1999, 52(2): 195-212.

[10]冯秀亮, 高志敏, 杨春荣, 雷安民, 樊敬庄, 窦忠英. 由猪囊胚内细胞团分离胚胎干细胞的研究. 西北农林科技大学学报, 2003, 31(3): 5-10.

Feng X L, Gao Z M, Yang C R, Lei A M, Fan J Z, Dou Z Y. Isolation of embryonic stem cells from inner cell mass of porcine blastocysts. Jourhal of Northwest A & F University Culture, 2003, 31(3): 5-10. (in Chinese)

[11]董晓, 王占贺, 王端云, 郑行. 猪胚胎干细胞培养、分离和传代. 农业生物技术学报, 2003, 11(3): 263-267.

Dong X, Wang Z H, Wang D Y, Zheng X. Culture, isolation and passage of porcine embryonic stem cells. Journal of Agricultural Biotechnology, 2003, 11(3): 263-267. (in Chinese)

[12]Shim S, Han D, Yang J, Lee B, Kim S, Shim H, Lee H, Derivation of embryonic germ cells from post migratory primordial germ cells, and methylation analysis of their imprinted genes by bisulfite genomic sequencing. Molecules and Cells, 2008, 25(3), 358-367.

[13]Piedrahita J, Moore C, Oetama B, Lee C K, Scales N, Jagdeece R, Bazer F, Ott T. Generation of transgenic porcine chimeras using primordial germ cell-derived colonies. Biology of Reproduction, 1998, 58 (5), 1321-1329.

[14]Tsung H, Du Z, Rui R, Li X, Bao L, Wu J, Bao S, Yao Z. The culture and establishment of embryonic germ (EG) cell lines from Chinese mini swine. Cell Research, 2003, 13 (3): 195-202.

[15] Petkov S G, Marks H, Klein T, Garci R S, Gao Y, Stunnenberg H, Hyttel P. In vitro culture and characterization of putative porcine embryonic germ cells derived from domestic breeds and Yucatan mini pig embryos at Days 20-24 of gestation. Stem Cell Research, 2011, 6(3): 226-237.

[16]况玲, 冯书堂, 余四九, 牟玉莲, 张勇. 猪胚胎多能性干细胞的分离培养. 中国兽医科技, 2004, 34(7): 55-59.

Kuang L, Feng S T, Yu S J, Mu Y L, Zhang Y. Isolation of pluripotent stem cells from cultured porcine germ cells, Chinese Journal of Veterinary Science and Technology, 2004, 34(7): 55-59. (in Chinese)

[17] Pei D Q, Esteban M A, Xu J Y. Generation of induced pluripotent stem cell lines from Tibetan miniature pig. The Journal of Biological Technology, 2009, 284(26): 17634-17640.

[18] Ezashia T, Telugua B P V L, Alexenkoa A P, Sachdevb S, Sinhaa S, Robertsa R M. Derivation of induced pluripotent stem cells from pig somatic cells. Proceeding of National Academy Science of United State of America, 2009, 106(27): 10993-10998.

[19]Eiselleova L, Peterkova I, Slaninova I, Neradjl J, Hampl A, Dvorak P. Comparative study of mouse and human feeder cells for human embryonic stem cells. International Journal of Developmental Biology, 2008, 52(4): 353-363.

[20]Lee C K, Piedrahita J A. Effects of growth factors and feeder cells on porcine primordial germ cells in vitro. Cloning, 2000, 2(4): 197-205.

[21]Spiller C, Wilhelm D, Koopman P. Cell cycle analysis of fetal germ cells during sex differentiation in mice. Biology of the Cell, 2009, 101(3): 587-598.

[22]Western P, Miles D, Van Den Bergen J, Burton M, Sinclair A. Dynamic regulation of mitotic arrest in fetal male germ cells. Stem Cells, 2008, 26(3): 339-347.

[23] Ewen K A, Koopmana P. Mouse germ cell development: From specification to sex determination. Molecular and Cellular Endocrinology, 2009, 323(1): 76-93.

[24]Sun Y, Li H, Yang H. Mechanisms controlling embryonic stem cell self-renewal and differentiation. Critical Reviews in Eukaryotic Gene Expression, 2006, 16 (3): 211-231.

[25]Ying Q L, Stavridis M, Griffiths D. Conversion of embryonic stemcells into neuroectodermal precursors in adherent monoculture. Nature Biotechnology, 2003, 21 (2): 183-186.

[26]Hua B Y, Weicka J P, Yub J Y. Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proceeding of National Academy Science of United State of America, 2010, 107(9): 4335-4340.

[27]Kriks S, Shim J W, Piao J H. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature, 2011, 480: 547-551.

[28]Chambers S M, Fasano C A, Papapetrou E P. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnology, 2009, 27(3): 275-280.