摘要
近年来，作为精子和卵子的祖细胞的原始生殖细胞(PGCs)已成为另一种可选择的多能性干细胞。来源于PGCs的干细胞称胚胎生殖细胞。迄今为止仅在小鼠上获得PGCs来源的真正意义上的胚胎干细胞(ES cells)，即具有参与生殖系传递能力的ES细胞。
取孵化5.5～6.5天含PGCs的鸡胚生殖腺及其周围组织，经0.25％胰酶+0.02％EDTA消化后，接种于24孔培养板内。加入新鲜的EG细胞培养液(含DMEM、胎牛血清、鸡血清、β-巯基乙醇、L-谷氨酰胺、HEPES、鸡胚浸出液以及细胞因子等成分)培养24小时以后，PGCs开始部分贴附于共培养的生殖原基细胞上。培养3～4天的PGCs大多已转变为由2～20细胞构成的集落，其中包括从PGCs群集或细胞本身增殖所形成的两种集落类型。培养至5～7天，类EG克隆数目明显增加。类EG细胞克隆呈规则的集落状生长，多层分布，且有明显的花纹。克隆内的细胞间并不紧密排列，因此不难看出克隆内的单个细胞。这与经典的小鼠胚胎生殖细胞的形态有所不同。
原代培养时，我们采用PGCs与相同来源的生殖原基细胞共培养的方式，效果良好。继代培养时，经过反复实验比较鸡胚原代成纤维细胞饲养层(PCEF)、小鼠原代成纤维细胞饲养层(PMEF)、SNL细胞饲养层等三种不同饲养层的作用效果，最终发现以鸡胚原代成纤维细胞制作饲养层同时添加各种生长因子最适合鸡类EG细胞的生长，但PCEF与PMEF之间的差异不显著。本研究中，通过分组添加不同生长因子，结果发现，培养初期不同的分组在培养过程中出现的类EG集落数相差不明显。而随着培养时间的延长，添加LIF、bFGF、SCF、及IGF-1组表现的优势越来越明显，对促进类EG集落的持续增多效果最佳。四种生长因子的添加量分别为：100IU／ml，10ng／m1，10ng／ml，10ng／ml。
在鸡类EG细胞的传代过程中，采用0.25％胰酶与0.02％EDTA的联合作用将集落离散收到了很好的效果。胰酶和EDTA的协同作用，缩短了消化时间，同时降低了消化液对细胞的损伤作用。除酶的作用以外，外源性物质的添加、血清的更换、温度和PH值的改变等因素对鸡类EG细胞的分离培养均有重要的影响。
研究中分离得到的鸡类EG细胞经PAS染色后呈深紫红色，而鸡胚成纤维细胞并不着色。将消化分散的类EG细胞在无分化抑制因子的类EG培养基中重悬培养，3～4天后，可见部分细胞团形成简单的胚体。在饲养层上生长的类EG细胞集落，在去除抑制分化因子的条件下培养，长时间不传代，可见细胞生长缓慢，自发分
山东农业大学硕士研究生论文（2003）
化，首先表现为失去原有的形态，进而与周围细胞界限不清，集落外围的细胞与饲
养层连成一片。最后将传至3代、4代的类EG细胞集落离散后制作嵌合体，得到了
一只毛色嵌合体鸡。
本研究分离得到的类EG细胞经形态学鉴定、PAS染色、体外分化实验和嵌合
体鸡的制作等实验证明其具有胚胎干细胞的诸多类似特性。类EG细胞所具有的多
能性、定向整合及较高的嵌合能力，对高效培育鸡的新品种有重大的意义。
Recently, primordial germ cells(PGCs), which are the progenitors of the sperm or EGg cells, have provided an alternative source of pluripotent stem cells. The stem cells derived from PGCs are called embryonic germ cells(EG cells). To date, EG cells with proven germ-line transmission have been completely established only in the mouse with embryonic stem cells(ES cells).
Gonadal primordial germ cells were got by isolating embryonic gonads and surrounding tissues from chicken embryos(5.5-6.5 days of incubation) and dissociating the tissues by standard trypsinization procedure(0.25%Trypin+0.02%EDTA). The EG cell culture media consisting of DMEM medium supplemented with FBS, chicken serum, beta-mercaptoethanol, L-glutamine. HEPES, chicken embryonic extract and cytokines etc. After 24 hours culture, the isolated PGCs were selectively attached on the gonadal stromall cells in the plates. However, at 3~4 days primary culture, almost all the PGCs were formed of 2~20 cell mass. After 5~7 days, the numbers of putative EG clonies increased distinctly. These clonies were uniformly round, multi-layered and well delineated. The cells did not pack strongly together in small clumps and it was not difficult to discern the individual component cells. This morphology was slightly different from that of mouse EG cells.
At primary culture, PGCs co-cultured with their gonadal stromall cells were well grown . When subculture, we used primary chicken embryonic fibroblast (PCEF), primary mice embryonic fibroblast (PMEF) and SNL cells to make feeder cells. Forward research founded that the PCEF cells were the most suitable for the growth of putative EG cells when having various cytokines. In this study, the putative EG cell culture media was supplemented with LIF, bFGF, SCF and IGF-1. As a result, the ideal
dose of LIF, bFGF, SCF and IGF-1 was: 100IU/ml,10ng/ml, 10ng/ml, 10ng/ml. In addition, there were still many other factors were influencing on the culture of putative EG cells such as the density of FBS, the cell-dispersed liquid, the temperature and PH of the culture environments etc.
In this study, the chicken putative EG cells continued to- be PAS positive and manifested some typical morphology. Under suspension culture without cytokines, the putative EG clonies could be induced to form simple embryoid bodies. After long-time culture, these bodies began to differentiated into various cell types. Moreover, chimeras when injecting of the cultivated cells into receptor's x stage blastdermal also showed that the cells were some pluripotent. These study may be useful to the production of transgenic chickens by genomic modification.
引文
1. Evans,M.J., M.H.Kaufman, 1981 Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154～156.
2. Resnick JL, Bixler LS, Cheng L, et al. 1992, Long-term proliferation of mouse primordial germ cells in culture. Nature, 359(6395):550
3. MastuiY, ZseboK. 1992 Drivation of pluripotential embryonic stem cells from murine primordial germ cells in culture[J].Cell, 70:841.
4．许新，丛笑倩，严缘昌．1999 小鼠原生殖细胞建系过程及其分化特性的研究，实验生物学报，32(3)：251～263
5. Stewart C.L., Gadil., Bhatt H. 1994 Stem cells from primordial germ cells can reeter the germ line. Der. Biol., 161(2):626～628
6. Mitani T, Takahashi N, kawase E et al. 1994 Proliferation of alkaline phosphotase-positive cells from cultured primordial germ cells in rat. Theriogenology, 41:336
7. Piedrahita, Jorge A, Karen moore, Betty Oetama et al. 1998 Generation of transgenic porcine chimeras usine primordial germ cell derived colonies. Biology of Reproduction, 58:1321～1329
8. Shim H, Gutierrez-Adan A,Chen LR et al. 1997 Isolation of pluripotent stem cells from cultured porcine primordial germ cells. Biology of Reproduction, 57:1089～1095
9. Shim H, Anderson G.B. 1998 In vitro survival and proliferation of porcine germ cells. Theriogenology, 49:521～528
10. Shamblott M.J., Axelman J., Wang S. et al. 1998 Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc. Natl. Sci. USA, 95:13726～13737
11．华进联，窦忠英，李松．2001自PGCs分离克隆哺乳动物ES细胞的研究进展，农业生物技术学报，9(1)：97～102
12. Park,T.S, J.Y.Han, 2000 Derivation and characterization of pluripotent embryonic germ cells in chicken. Mol Reprod and Deve. 56:475～482.
13. Elliot, T.S., 2000 The avian primordial germ cell: An enigma of germline evolution,development and immortality. Avian and Poultry Biology Reviews. 11(4): 173～282.
14. Meyer D.B. 1964 The migration of primodial germ cells in the chick embryo[J]. Dev, 10:154～190
15. NakamuraM,Kuwanal,MiyaymaY, etal. 1988 Extra gonadal distribution of primordial germ cells in the early chick embryo[J]. Anat Rec, 222:90～94
16. TsunodaY, TokunagaT,ImaiH. 1989 Nuclear transplantation of male primordial germ cells in the mouse[J]. Development,, 107:407～411
17. Fujimoto T, Ukeshina A, Kiyofuji R. 1976b The oyigin,migration and morphology of the primordial germ cells in chicke mbryo. Anat Res, 185:139～154
18. Karagenc,L., Y.Cinnamon, M.Ginsburg, and J.N.Petitte, 1996 Origin of primordial germ cells in the prestreak chick embryo. Dev Gene. 19:290～301.
19. Kuwana, T., Y.Miyamama, Y. Kojiwara, and T.Rujimoto, 1987 Behavior of chick primordial germ cells moving toward gonadal primordium in vitro:Scanning electron microscopic study. Anat.Rec. 219:164～170.
20. Rudnicki M A, McBurney M.W. 1987 Cell culture methods and indution of embryo nalcarcinoma cell lines[C].Teratocarcinoma and embryonic stem cell[A]. Oxford: Robertson edited, 19～49
21．刘荣秀，1990 鸟类原生殖细胞的研究Ⅰ：鸡胚生殖新月区的原生殖细胞超微结构的观察动物学报，36(2)：114～118
22. Clawson R.C., Domm L.V. 1963a Developmental changesin glycogen content of primodial germ cells in chick embryo[J]. Proc Soc Exptl Biol Med, 112:533～537
23. Kuwana T. 1987 Behavile of chick primordial germ cells moving toward gonadal primordium in vitro:scanning lectron microscopic study[J]. The Anat Rec, 219:164～170
24. Eyal-Giladi H, Ginsburg M, Farbarov A. 1981 Avian primordial germ cell are of epiblastic origin [J].JE mbrryol Exp Morph, 65:13
25. GinsburgM,Eyal-GiladiH. 1986 Temporal and spatial aspects of the gradual migration of primordial germ cells from the epiblast into the germinal crescent in the avain embryo[J]. J Embryol Exp Morphol, 95:53～71
26. Nieuwkoop, P.D. and Sutasurya, L. A. 1979 Primordial germ cells in the Chordates. Cambridge Uni. Press
27. KuwanaT. 1987 Behavile of chick primordial germ cells moving toward gonadal primordium in vitro: scanning electron microscopic study[J]. The Anat Rec, 219:164～170
28．李碧春，陈国宏 2001 禽类原始生殖细胞与转基因研究进展．国外畜牧科技 28(3)：26～30．
29. UkeshimaAMK,FujimotoT. 1987 Relationship between genital vidge formation and settlement site of primordial germ cells in chick embryos[J]. Anat Rec, 219:311～314
30. Petitte,J.N., M.F.Clark, and R.J.Etches., 1991 Assessment of functional gametes on chickens after tranfer of primordial germ cells. J.Reprod.Fert. 92:225～229.
31. Abdel-MalekET. 1950 Early development of the urinogenital system in the chick[J]. JMorphol, 86:599～626
32. Wentworth,B.C., H.Tsai, J.H.Hallett, D.S.Gonzales, and S.G.Rajcic, 1989 Manipulation of avian primordial germ cells and gonadal differentiation. Poultry Sci. 68:999～1010. 1:107～116.
33. Hajji,K, C.Martin, A.Perramon, and F.Dirterien-lievre, 1988 Sexual phenotype of avian chimeric gonads iwith germinal and stromal cells of the opposite genetic sexes.
Biol.Struct.and Morpho.
34. Allioili N, Thomas J, Chebloune Y, Nigon V, Verdier G, 1994 Use of retrovial vectors to introduce and express the beta-galatosidase markergene in cultured chicken primordial germ cells. Der Biol, 165:30～37
35. Chang,I.K., A.Yoshiki, M.Kusakabe, A.Tajima, T.Chikamune, M.Natio, and T.Ohno, 1995 Germ line chimera produced by transfer of cultured chick primordial germ cells. Cell Biol.Int. 19(7):569～576.
36. Chang,I.K., Jeong,D.K., 1997 Production of germline chimeric chickens by transfer of cultured primordial germ cells. Cell.Biol.Int., 21:495～499.
37. Jae Yong Han, Tae Sub Park, Yeong Ho Hong, Dong Kee Jeong, Jin Nam Kim, Ki Dong Kim and Jeong Mook Lim, 2002 Production of germline chimeras by transfer of chicken gonadal primordial germ cells maintained in vitro for an extended period. Theriogenology, 58:1531～1539
38．邹清雁，张淑莲，郑曲波，孔祥平，肖荣，张宜俊 2000 鸡胚胎干细胞饲养层培养体系的建立．上海实验动物科学 20(4)：206～209．
39．杜立新，李善刚 1999 鸡胚胎体外培养和嵌合体制作的研究．第十次全国动物遗传育种学术讨论会论文集．
40．安静，杜立新 2002 鸡胚胎干细胞的分离、培养及鉴定．山东农业大学硕士研究生毕业论文
41．李赞东，刘春海，黄劲松，沙金，危华，孙明军等．2002 鸡鸭异种间嵌合体的制备．动物学报，48(4)：543～548
42．李碧春，陈国宏，2001 禽类原始生殖细胞与转基因研究进展．国外畜牧科技 28(3)：26～30．
43．尚克刚，胡新立，李子玉，王学庆，刘爱民，孟国良，童英 1994 饲养层对维持新建ES细胞系的影响．北京大学学报(自然科学版) 30(4)：500～508．
44. De Felici M and Mclaren A. 1982 Isolation of mouse primordial germ cells. Exp Cell Res, 142:476～482
45. De Felici M and Mclaren A. 1983 In vitro culture of mouse primordial germ cells. Exp Cell Res, 144:417～427
46. De Felici M, Pesce M, Giustiniani Q, Di Carlo. 1998 In vitro adhesiveness of mouse primordial germ cells to cellular and extracellular matrix component substrata. Microsc Res Tech, 43(3):258～264
47．周振明，桑润滋，李俊杰 2001 胚胎干细胞分离合克隆影响因素的研究进展．黄牛杂志 27(4)：30～34．
48. Ware LM and Axelrad AA. 1972 Inherited resistance to N-and B-tropic murine leukemia viruses in vitro: Evidence he cogenic mouse strains SIM and SIM.R differ at the FV-1 locus. Virology, 50:339～348
49. Mather JP. 1980 The establishment and characterization of two distinct mouse testicular epithelial cell lines. Biol Rprod, 23:243～252
50．韩嵘，尚克刚 2000 具有高效种系嵌合能力的小鼠胚胎干细胞系的建立和特异标记(博士论文)
51. Matin G R. 1981 Factor influencing the isolation of murine embryonic stem cells. Proc Natl Acad Sci USA, 78:7634
52. Sims M..M. andd N.L. First. 1993 Production of fetuses from totipotent cultured bovine inner cell mass cells. Development Biology, 121:1～9
53. Donovan PJ. 1994 Growth factor rEGulation of mouse primordial germ cell development. Current Topics in Developmental Biology, 29:189～225
54. Uchida M, Tokunaga T, Niwa K et al. 1995 Effect of feeder cells and growth factrs on the proliferation of mouse primordial germ cells. Theriogenology, 44(1):9～16
55. De Felici F, Dolci S, Pesce M et al. 1993 Proliferation of mouse primordial germ cells in vitro: a key role of cAMP Devel Biol, 175(1):277～280
56. Dolci S, Williams DE, Ernst M et al. 1991 Requirement for mast cell growth factor for primordial germ cell survival in culture. Nature, 352:809～811
57．司徒镇强，吴军正．1996 细胞培养．世界图书出版公司
58. Tillmann S.M. and Meinecke.B. 1996 Isolation of ES-like cell lines from bovine and caprine preimplantation embryos. J.Anim.Breed Genet, 113:413～426
59．鄂征主编．1986 组织培养技术(修正本)，人民卫生出版社
60．叶鑫生，等．2000 干细胞与发育生物学[M] 北京：军事医学科学出版社
61．邱观婷，黄冰，唐仕波，胡洁，刘志雄 2000 探讨保持胚胎干细胞全能性的体外培养条件．中山医科大学学报 21(2)：100～103．
62．孟国良，汤富酬，腾路，尚克刚 2001 小鼠胚胎干细胞(ES细胞)建系和维持过程中的问题及对策．遗传 23(3)：292～294．
63．尚克刚，胡新立，李子玉．1994 饲养层对维持新建ES细胞系的影响．北京大学学报(自然科学版)，30(4)：500～507
64. Uator Y, Tsunoda. 1993 Totipotency and pluripency of embryonic nuclei in the mouse. Molecular Reproduction and Development, 36:276～278
65．李松，窦忠英．2001 从原生殖细胞中分离克隆牛胚胎干细胞．西北农业大学硕士研究生毕业论文
66．丛笑倩，姚鑫．1987 小鼠胚胎干细胞建系过程的核型及特性分析．实验生物学报，20：237～251
67. Matsui,Y., K.Zsebo, and B.L.M.Hogan, 1992 Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell Sep 4 70:841～847.
68. Nakagawa,S Saburi,S Yamanouchi,K Tojo,H Tachi,C 2000 In vitro studiES on PGC or PGC-like cells in cultured yolk sac cells andembryonic stem cells of the mouse.
ArchivES of histology and cytology, 63(3): 229-241
69. Divine,J.K.,N.Flake, 2000 Expression of a novel antigen, EEM-1,in the mouse embryo and embryonic stem cell -derived embryoid bodies. Decelopmental Dynamics 218:207～211.
70. Thomson,J.A., J.S.Odorico, 2000 Human embryonic stem cell and embryonic germ cell lines. Tibtech Feb 18:53～57.
71. Pera,M.F., B.Reubinoff, A.Trounson, 2000 Human embryonic stem cells. Jour of Cell Sci. 113:5～10.
72．张玲，贾红梅，贾心善 2000 胚胎干细胞体外分化研究的最新进展．日本医学介绍 21(11)：524～525．
73. Roger A.Pedersen, 1994 Studies of in vitro differentiation with embryonic stem cells. Reprod.Fertil.Dev. 6:543～552.
74．潘兴华，郭坤元，陈系古，陈永乐，陆家海 2000 胚胎干细胞的生物学特性及体外诱导分化．生命科学研究 4(2) (Suppl)：48～54．
75. Munsie,M.J.,.E.Michalska, C.M.O'Brien, A.O.Trounson, M.F.Pera, and P.S.Mounfford, 2000 Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei. Current Biology. 10(16):989～992.
76. Bradley, A., M.Evans, M.H.Kaufman, and A.E.Robertson, 1984 Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature May 309(17):255～256.
77. Gearhart J, 1998 New potential for human embryonic stem cells. Science, 282:1061～1062
78. Marshall E, 1998 A versatile cell line raises scientific hopes, lEGal questions. Science, 282:1014～1015
79. Buehr M. 1997 The primordial germ cells of mammals: some current perspectives. Exp Cell Res, 232:194～207
80. Natio,M, E.Sasaki, and M.Ohtaki, 1994 Introduction of exogenous DNA into Somatic and germ cells of chickens by microinjection into the germinal disc of fertilized ova. Mol.Reprod.Dev. Feb 37(2): 167～171.
81. Stewart,C.L., I.Gadi, and H.Bhatt, 1994 Stem cells from primordial germ calls can reenter the germ line. Developmental Biology. 161:626～628.
82. Etches,R.J., R.S.Carasience, M.E.Clark, R.A.Fraser, A.Toner, and A.MLVerrinder Gibbins, 1993 Chimeric chickens and their use in manipulation of the chickens genome. Poultry Science. 73:882～889.
83. Kuwana T, Rogulska T. 1999 Migratory mechanisms of chick primordial germ cells toward gonadal anlage. Cell Mol Biol. 45(5):725
84. Pain,B., P. Chenevier, and J.Samarut 1999 Chicken embryonic stem cells and transgenic stratEGies. Cells Tissues Organs. 165:212～219.
85. Simdiss,K., Y. Li, G.Luke, N.Page, and L.Vick, 1993 The production of transgenic
birds from primordial germ cell manipulated embryos. Journal of Endocrinology Ltd, Bristol. 367～380.
86．杨威，陈岩，朱大海．2002 禽类胚胎干细胞的研究．高技术通讯，12(1)：102-105
87．张学明，李德雪，赖良学，李子义，文兴豪，杜惜明，曾发贵 1999 小鼠胚胎成纤维细胞的分离合饲养层细胞的制备．黑龙江畜牧兽医 9：5～6．
88．王杏利，窦忠英 1999 小鼠胎儿成纤维细胞的分离培养及饲养层制备．陕西农业科学 6：17～19．
89．高舒平，时伟红，秦英，董婉维，王太一 2000 用于胚胎干细胞分离培养的饲养层的制作．中国实验动物学杂志 10(2)：78～81．
90．杨奇，史远刚，窦忠英 2001 影响小鼠胎儿成纤维细胞饲养层的因素．动物医学进展 22(4)：45～47．
91．薛庆善主编，2001 体外培养的原理与技术．科学出版社
92. Well, D.N., Mewhir, J., Hooper, M.L., Willmut, I., 1996 Factor influencing the isolation of murine embryonic stem cells. Theriogenololgy, 35:293～298
93. Rathjen, P.D., Nichols, J., Toth, S,. Edwards, D.R., Heath, J.K. and Smith, A.G., 1990 Developmentally programmed induction of differentiation inhibiting activity and the control of stem cell populations. Genes. Dev., 4:2308～2318
94．常万存，窦忠英，高志敏等 2000 人胚胎成纤维细胞的冷冻保存．西北农业学报，9(1)：6～9．
95．马新武 2000 哺乳动物胚胎冷冻原理及研究进展．动物医学进展，21(4)112～115．
96．韩毅冰，于康震，周琦，秦鹏春 1996 鸡胚血液中原始生殖细胞的分离及其培养的研究生物技术，6(2)：11～13
97．刘春海，李赞东，黄劲松，沙金，危华，赵晨，孙明军 2001 鸡胚原始生殖细胞的分离和鸡鸭嵌合体的制备研究河北农业大学学报，24(3)：62～66
98．李碧春，陈国宏，赵东伟，钱菊汾 2001 鸡胚血液中 PGCs 的分离及原代培养 中国兽医学报，21(4)：370～372
99．张天荫 1993 鸟类的原生殖细胞细胞 生物学杂志，15(2)：49～53
100. LeeCK,ScalesN,NewtonG,etal.Isolationandinitialcharacterizationofprimordialgermcel 1(PGC)-derivedcellsfromgoat, rabbitandrats[J].Theriogenotogy, 1998,49,388(abstract)
101．常万存 窦忠英 马鸿飞等，1998 原始生殖细胞的人类胚胎干细胞克隆．西北农业大学学报，26(6)：105～108
102. Maova, K,. Bachvaove, R.E, 1991 Expression of c-kit encoded at the W locus of mice in developing primordial germ cells and presumptive melanblast. Develop. Biol., 146:312～324
103. Suemori,H., N.Nakatsuji, 1987 Establishment of the embryo-derived stem(ES)cell lines from mouse blastocysts: Effects of the feeder cell layer. Develop. Growth and
Differ. 29(2):133～139.
104. Pease,S., P. Braghetta, K.Gearing, D.Grail, and R.L.Williams, 1990 Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor(LIF). Developmental Biology. 141:344～352.
105. Cheng,L., D.P.Gearing, L.S.White, D.L.Compton, K.Schooley, and P.J.Donovan, 1994 Role of leudimia inhibitory factor and its receptor on mouse primordial germ cell growth. Development 120:3145～3153.
106. Resnick,J.L., M.Ortiz, J.R.Keller, and P.J.Donovan, 1998 Role of fibroblast growth factors and their receptors in mouse primordial germ cell growth. Biology of Reproduction. 59:1224～1229.
107. Yan,W., J.Suominen, J.Toppari, 2000 Stem cell facter protects germ cells from apoptosis in vitro. Journal of Cell Science. 113:161～168.
108. Pesce,M., M.G.Farrace, M.Piacentini, S.Dola, and M.De Felici, 1993 Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis). Development 118:1089～1094.
109. Scotlandi K Avnet S Benini S Manara MC Serra M Cerisano V Perdichizzi SLollini PL De Giovanni C Landuzzi L Picci P 2002 ExprESsion of an IGF-I receptor dominant nEGative mutant inducES apoptosis, inhibits tumorigenES is and enhancES chemosensitivity in Ewing's sarcoma cells. International Journal of Cancer, 101(1):11～16
110. Leichthammer F, Baunack E, Brem G. 1990 Behavior of living primordial germ cells of livestock in vitro. Theriogenology, 33 (6): 1221～1230
111. Pesce M, Farrace MG, Piacentini M, et al. 1993 Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death(apoptosis). Dev, 118:1089～1094