小鼠MSCs多能性及其与颗粒细胞相互影响的研究
|
摘要
目的：
探索小鼠MSC具有多分化潜能的原因以及MSCs对卵巢GC的影响。
方法：
1、从小鼠骨髓中分离MSCs,并进行原代培养,分别于0、2、4、6、8、10天提取总RNA,RT-PCR检测多潜能标记基因和相关因子、三个胚层标记基因的表达情况。
2、利用分离、纯化的MSCs与GC,建立共培养体系。实验分3组：单独培养MSCs组、单独培养GC组及MSCs和GC共培养组。分别在4天和6天提取总RNA,RT-PCR检测卵巢内标记基因在GC和MSCs中的表达情况,以及共培养MSCs多潜能标记基因和相关因子及三个胚层基因的表达情况。并在3天和5天通过MTT法检测各组细胞的增殖情况。
结果：
小鼠MSCs表达多潜能性标记基因Oct-4、nanog和相关因子Klf4和c-Myc.三个胚层基因nestin、SM22α、CYP51均有表达。在共培养实验中,单独培养和共培养GC均有ZP1.ZP2.ZP3和FSHR的表达,但ZP3.FSHR和BMP-15表达量不同。MSCs可促进GC增殖。不同天数单独培养和共培养MSCs均未检测到卵巢内标记基因的表达,但多能性基因和相关因子表达量不同,三个胚层基因表达无变化。此外,GC可促进MSCs增殖。
结论：
贴壁筛选得到的小鼠MSCs表达多能性标记基因以及三个胚层标记基因。在共培养体系中,GC与MSCs之间可以相互促进增殖并影响基因的表达。
Objective:
To explore the cause of multi-differentiation potential of mouse bone marrow mesenchymal stem cell(MSCs) and the effect of MSCs on mouse ovarian granulosa cells (GC) in vitro co-cultured.
Methods:
MSCs were isolated from the bone of the adult mouse and cultured in vitro.Total RNA were extracted from the mouse bone marrow (Day 0) and cultured mouse MSCs at 2,4,6,8 and10 days. The pluripotency marker genes Oct-4 and nanog, pluripotency related transcription factors Klf4 and c-Myc and the marker genes of three embryonal layers, such as CYP51, SM22αand nestin, which are typical genes specific for endoderm, mesoderm and ectoderm respectively, were detected by RT-PCR.
MSCs and GC from mouse were cultured and purified in vitro using their adherent characteristics. A co-culture system was established by culturing MSCs in the Transwell insert and GC on the out plastic plates (6 wells). The cells were divided into 3 groups:alone cultured MSCs, alone cultured GC and MSCs co-cultured with GC. Total RNA were extracted from the cells of these 3 groups at the 4th day and the 6th day respectively. The ovarian marker genes mRNA were detected by RT-PCR. The pluripotency related genes and the three embryonal layers genes were also detected at the 4th day and the 6th days respectively. Cell proliferation of these 3 groups were detected by MTT assay after 3 days and 5 days respectively.
Results:
The mRNA of Oct-4, nanog, Klf4 and c-Myc were detected in mouse MSCs. Furthermore, in addition to SM22a, RT-PCR revealed the presence of mRNA for CYP51 and nestin during the primary culture of mouse MSCs. the mRNA of ZP1, ZP2 and ZP3, FSHR were detected in both alone cultured and co-cultured GC at the 4th day and the 6th day. But the mRNA of ZP3, FSHR and BMP-15 were different between co-cultured GC and alone cultured GC.The mouse MSCs promoted the proliferation of the mouse ovarian GC. No mRNA of ovarian characteristics genes were detected in alone cultured or co-cultured mouse MSCs. The mRNA of pluripotency marker genes and pluripotency related transcription factors were different between co-cultured MSCs and alone cultured MSCs, while the mRNA of genes of the three embryonal layers did not significantly change. In addition, the mouse ovarian GC promoted the proliferation of mouse MSCs.
Conclusion:
Mouse MSCs, which were isolated by adhesion purification, expressed the mRNA of the pluripotency marker genes and the marker genes of three embryonal layers. In vitro co-culture, the proliferation of the mouse ovarian GC and the mouse MSCs were promoted mutually and affected the quantity of mRNA for some genes.
探索小鼠MSC具有多分化潜能的原因以及MSCs对卵巢GC的影响。
方法：
1、从小鼠骨髓中分离MSCs,并进行原代培养,分别于0、2、4、6、8、10天提取总RNA,RT-PCR检测多潜能标记基因和相关因子、三个胚层标记基因的表达情况。
2、利用分离、纯化的MSCs与GC,建立共培养体系。实验分3组：单独培养MSCs组、单独培养GC组及MSCs和GC共培养组。分别在4天和6天提取总RNA,RT-PCR检测卵巢内标记基因在GC和MSCs中的表达情况,以及共培养MSCs多潜能标记基因和相关因子及三个胚层基因的表达情况。并在3天和5天通过MTT法检测各组细胞的增殖情况。
结果：
小鼠MSCs表达多潜能性标记基因Oct-4、nanog和相关因子Klf4和c-Myc.三个胚层基因nestin、SM22α、CYP51均有表达。在共培养实验中,单独培养和共培养GC均有ZP1.ZP2.ZP3和FSHR的表达,但ZP3.FSHR和BMP-15表达量不同。MSCs可促进GC增殖。不同天数单独培养和共培养MSCs均未检测到卵巢内标记基因的表达,但多能性基因和相关因子表达量不同,三个胚层基因表达无变化。此外,GC可促进MSCs增殖。
结论：
贴壁筛选得到的小鼠MSCs表达多能性标记基因以及三个胚层标记基因。在共培养体系中,GC与MSCs之间可以相互促进增殖并影响基因的表达。
Objective:
To explore the cause of multi-differentiation potential of mouse bone marrow mesenchymal stem cell(MSCs) and the effect of MSCs on mouse ovarian granulosa cells (GC) in vitro co-cultured.
Methods:
MSCs were isolated from the bone of the adult mouse and cultured in vitro.Total RNA were extracted from the mouse bone marrow (Day 0) and cultured mouse MSCs at 2,4,6,8 and10 days. The pluripotency marker genes Oct-4 and nanog, pluripotency related transcription factors Klf4 and c-Myc and the marker genes of three embryonal layers, such as CYP51, SM22αand nestin, which are typical genes specific for endoderm, mesoderm and ectoderm respectively, were detected by RT-PCR.
MSCs and GC from mouse were cultured and purified in vitro using their adherent characteristics. A co-culture system was established by culturing MSCs in the Transwell insert and GC on the out plastic plates (6 wells). The cells were divided into 3 groups:alone cultured MSCs, alone cultured GC and MSCs co-cultured with GC. Total RNA were extracted from the cells of these 3 groups at the 4th day and the 6th day respectively. The ovarian marker genes mRNA were detected by RT-PCR. The pluripotency related genes and the three embryonal layers genes were also detected at the 4th day and the 6th days respectively. Cell proliferation of these 3 groups were detected by MTT assay after 3 days and 5 days respectively.
Results:
The mRNA of Oct-4, nanog, Klf4 and c-Myc were detected in mouse MSCs. Furthermore, in addition to SM22a, RT-PCR revealed the presence of mRNA for CYP51 and nestin during the primary culture of mouse MSCs. the mRNA of ZP1, ZP2 and ZP3, FSHR were detected in both alone cultured and co-cultured GC at the 4th day and the 6th day. But the mRNA of ZP3, FSHR and BMP-15 were different between co-cultured GC and alone cultured GC.The mouse MSCs promoted the proliferation of the mouse ovarian GC. No mRNA of ovarian characteristics genes were detected in alone cultured or co-cultured mouse MSCs. The mRNA of pluripotency marker genes and pluripotency related transcription factors were different between co-cultured MSCs and alone cultured MSCs, while the mRNA of genes of the three embryonal layers did not significantly change. In addition, the mouse ovarian GC promoted the proliferation of mouse MSCs.
Conclusion:
Mouse MSCs, which were isolated by adhesion purification, expressed the mRNA of the pluripotency marker genes and the marker genes of three embryonal layers. In vitro co-culture, the proliferation of the mouse ovarian GC and the mouse MSCs were promoted mutually and affected the quantity of mRNA for some genes.
引文
[1]Friedenstein A J, Gorskaja J F, Kulagina N N. Fibroblast precursors in normal and irradiated mouse hematopoietic organs[J]. Exp Hematol,1976,4(5):267-272.
[2]Owen M E, Cave J, Joyner C J. Clonal analysis in vitro of osteogenic differentiation of marrow CFU-F [J]. J Cell Science,1987,87(5):731-738.
[3]Friedenstein A J, Chailakhyan R K, Gerasimov U V. Bone marrow osteogenic stem cells:in vitro cultivation and transplantation in diffusion chambers [J]. Cell Tissue Kinet,1987,20(3):263-272.
[4]David C C, Reiner C, Carla M et al. Rapid expansion of recycling stem cells in cultrues of plastic-adherent cells from human bone marrow[J]. Proc Natl Acad Sci, 2000,97(7):3213-3218.
[5]Yamaguehi M, Hirayama F, Kanai M,et al. Seru-free eoculture system for ex vivo expansion of human cord blood promotive progenitors and SCID mouse-reconstituting cells using human bone marrow primary stromal cells[J]. Exp Hematol,2001,29(2):174-182.
[6]Pittenger M F, Mackay A M, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells[J]. Science,1999,284(5411):143-147.
[7]Bruder S P, Jaiswal N, Hayneworth S E. Growth kinetics, self-renewal and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation [J]. J Cell Biochem,1997,64(2): 278-285.
[8]Vogel W, Grunerach F, Messam C A, et al. Heterogeneity among human bonemarrow-derived mesenchymal stem cells and neural progenitor cells[J]. Haematologica,2003,88(2):126-133.
[9]MackayAM, Beck S C, Murphy J M, et al. Chondrogentic differentiation of cultured human mesenchymal stem cells from marrow[J]. Tissue Eng,1998,4(4): 415-428.
[10]Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine[J]. MuscleNerve,1995,18(12): 1417-1426.
[11]Wang J S, Shum-Tim D, Galipeau J, et al. Marrow stromal cells for cellular cardiomyoplasty:feasibility and potential clinical advantages [J]. JThorc Cardiovasc Surg,2000,120(5):999-1006.
[12]Sanchez-Ramos J,Song S,Cardozo-Pelaez F,et al. Adult bone marrow s tromal cells differentiate into neural cells in vitro[J]. Exp Neurol,2000,164(2):247-256.
[13]Woodbury D,Schwarz E J, Prockop D J, et al. Adult rat and human bone marrow stromal cells differentiate into neurons[J]. J Neurosci Res,2000,61(4):364-370.
[14]Robert E, Schwarts, Morayma Reyes, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells[J]. J Clin Inves t, 2002,109(10):1291-1302.
[15]Zurita M, Vaquero J. Bone marrow s tromal cells can achieve cure of chronic paraplegic rats:functional and morphological outcome one year after transplantation[J]. Neuroscience Letters,2006,402(10):51-56.
[16]Kopen G C, Prockop D J, Phinney DGMarrow stromalcellsmigrate throughout fore brain and cerebellum, and they dif-ferentiate into astrocytes after injection into neonatalmous brains[J]. Proc NatlAcad SciUSA,1999,96(19):107-111.
[17]Theise N D,Badve S, Saxena R, et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation[J]. Hepatology,2000, 31(1):235-240.
[18]Ferrari G, Angelis GC, Coletta M, et al. Mulscle regeneration by bone marrow-derived myogenic progenitors[J]. Science,1998,279(5356):1528-1530.
[19]Quarto R, Mastrogiacomo M, Cancedda R, et al. Repair of large bone defects with the use of autologous bone marrow stromal cell[J]. New Engl J Med,2001, 344(5):385-386.
[20]Pesce M, Scholer H R. Oct4:control of totipotency and germline determination[J]. Mol Reprod Dev,2000,55(4):452-457.
[21]Niwa H, Miyazaki J-I, Smith A G Quantitative expression of oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells[J]. Nat Genet,2000,24(4): 372-376.
[22]Hart A H, Hartley L, Ibrahim M, et al. Identification, cloning and expression analysis of the pluripotency promoting Nanog genes in mouse and human[J]. Developmental Dynamics,2004,230(1):187-198.
[23]Calaleri F, Scholer H R. Nanog:a new recruit to the embryonic stem cell orchestra[J]. Cell,2003,113(5):551-552.
[24]Wang S H, Tsai M S, Chiang M F, et al. A novel NK typehomeobox gene, ENK (early embryo specific NK), preferentiallyexpressed in embryonic stem cells[J]. Gene Expression Patterns,2003,3(1):99-103.
[25]Pan G J, Pei D Q. Identification of two distinct transactivation domains in the pluripotency sustaining factor nanog[J]. Cell Research,2003,13(6):499-502.
[26]Hart A H, Hartley L, Ibrahim M, et al. Identification, cloning and expression analysis of the pluripotency promoting Nanog genes in mouse and human[J]. Dev Dyn,2004,230(1):187-198.
[27]Liu N, Lu M. The signal transduction pathways and molecules for ES cells self-renewal[J]. Chinese Science Bulletin,2005,50(8):721-726.
[28]Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J]. Cell,2006,126(4): 663-676.
[29]Yet S F, McA'Nulty M M, Folta SC, et al. Human EZF,a Kruppel-like zinc finger protein is expressed in vascular Endothelial cell sandcontain stranscriptional activationand repression domains[J]. J Biol Chem,1998,273(2):1026-1031.
[30]Evans P M, Zhang W, Chen X, et al. Kruppel-like factor 4 is acetylated by p300 and regulates gene transcription via modulation of histone acetylation[J]. J Biol Chem, 2007,282(47):33994-4002.
[31]Cartwright P, McLean C, Sheppard A, et al. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism[J]. Development, 2005,132(5):885-889.
[32]Li Y, McClintick J, Zhong L, et al. Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4[J]. Blood,2005,105(2):635-637.
[33]Lendahl U, Zimmerman L B,Mckay R. CNS stem cells express anew class of intermediate filament protein[J]. Cell,1990,60(4):585-595.
[34]Solway J, Forsythe S M, Halayko A J, et al. Transcriptional regulation of smooth muscle contractile apparatus expression[J]. Am J Respir Crit CareMed,1998,158(5): 100-108.
[35]Debeljak N, Hovrat S, Vouk K, et al. Characterization of the mouse lanosterol 14alpha-demethylase(CYP51), a new member of the evolutionarily most conserved cytoehorme P450 a family[J]. Arch Biochem BioPhys,2000,379(1),37-45.
[36]靳玉甫,覃爱平.卵巢颗粒细胞P450芳香化酶研究进[J].广西医科大学学报,2008,25(6)：972-974.
[37]Sommersberg B, Bulling A, Salzer U, et al. Gap junction communication and connexin 43 gene expression in a rat granulosa cell line:regulation by follicle-stimulating hormone[J]. Biol Reprod,2000,63 (6):1661-1668.
[38]Havelock J C, Rainey W E, Carr B R. Ovarian granulosa cell lines[J]. Mol Cell Endocrinol,2004,228(1-2):67-78.
[39]杨增明,孙青原,夏国良.生殖生物学[M].北京：科学出版社,2005.
[40]卢翠玲,杨巍,胡召元等.颗粒细胞的增殖分化及其在卵泡发育中的作用[J].科学通报,2005,50(21)：2341-2347.
[41]Hunter M H, Sterrett J J. Polycystic ovary syndrome:it's not just infertility [J]. Am Fam Physician,2000,62(5):1079-1090.
[42]Richards J S, Russell D L, Ochsner S, et al. Ovulation:new dimensions and new regulators of the inflammatory-like response[J]. Annu Rev Physiol,2002,64:69-92.
[43]徐晨,轴座民.生殖生物学理论与实践[M].上海：上海科学技术文献出版社,2005.
[44]Zheng W, Magid M S, Kramer E E. Follicle-Stimulating Hormone Receptor Is Expressed in Human Ovarian Surface Epithelium and Fallopian Tube[J]. American Journal of Pathology,1996,148(1):47-53.
[45]Prasad S V, Skinner S M, Carino C, et al. Structure and Function of the Proteins of the Mammalian Zona pellucida, Aachen[J]. Cells Tissues Organs,2000,166(2): 148-164.
[46]张鹏,石玉华,陈子江.生长分化因子-9及生长分化因子9B/骨形态发生蛋白-15与卵巢功能[J].生殖与避孕,2007,27(3)：204-208.
[47]Aaltonen J, Laitinen M P, Vuojolainen K, et al. Human growth differentiation factor 9(GDF-9)and its novel homolog GDF-9B are expressed in oocytes during early folliculogenesis[J]. J Clin Endocrinol Metab,1999,84(8):2744-2750.
[48]Moore R K, Otsuka F, Shimasaki S. Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells[J]. J Biol Chem,2003,278(1):304-310.
[49]Johnson J, Bagley J, Skazni k-Wikiel M, et al. Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood[J]. Cell,2005,122(2):303-315.
[50]贺卫卫,谢琪璇,秦俊文等.骨髓间充质干细胞对损伤卵巢的修复作用[J].暨南大学学报,2010,31(2)：120-124.
[51]Fu X, He Y, Xie C, et al. Bone marrow mesenchymal stem cell transplantation improves ovarian function and structure in rats with chemotherapy-induced ovarian damage[J]. Cytotherapy,2008,10(4):353-363.
[52]Lamoury F M J, Croitoru-lamoury J, Brew B J. Undifferentiated mouse mesenchymal stem cells spontaneously express neural and stem cell markers Oct-4 and Rex-1[J]. Cytotherapy,2006,8(3):228-242.
[53]Tropel P, Noel D, PlatetN, et al. Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow[J]. Exp Cell Res,2004,295(2):395-406.
[54]Hart A H, Hartley L, Ibrahim M, et al. Identification, cloning and expression analysis of the pluripotency promoting nanog genes in mouse and human[J]. Developmental Dynamics,2004,230(1):187-198.
[55]Esposito MT, DiNoto R, Mirabelli P, et al. Culture conditions allow selection of different mesenchymal progenitors from adult mouse bone marrow[J]. Tissue Engineering Part A,2009,15(9):2525-2536.
[56]Woodbury D, Reynolds K, Black I B. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal,and mesodermal genes prior to neurogenesis[J]. J Neurosci Res,2002,96(6):908-917.
[57]Ji K H, Xiong J, Hu K M, et al. Simultaneous expression of Oct4 and genes of three germ layers in single cell-derived multipotent adult progenitor cells[J]. Ann Hematol,2008,87(6):431-438.
[58]Mackenzie S M, McLaughlin E A, Perkins H D, et al. Immunocontraceptive effects on female rabbits infected with recombinant myxoma virus expressing rabbit ZP2 or ZP3[J]. Biol Reprod,2006,74(3):511-21.
[59]Williams Z, Litscher E S, Jovine L, et al. Polypeptide encoded by mouse ZP3 exon-7 is necessary and sufficient for binding of mouse sperm in vitro[J]. J Cell Physiol,2006,207(1):30-39.
[60]Tang J, Xie Q X, Pan S P, et al. Expression of recombinant human zona pellucida-3 protein(rhZP3)in Pichia pastoris[J]. Sheng WuGong Cheng Xue Bao, 2003,19(6):758-762.
[61]Naz RK, Rajesh C. Gene knockouts that cause female infertility:search for novel contraceptive targets[J]. Front Biosci.2005,10:2447-2459.
[62]Mannikko M, Tormala RM, Tuuri T. Association between sequence variations in genes encoding human zona pellucida glycoproteins and fertilization failure in IVF[J].Hum Reprod,2005,20(6):1578-1585.
[63]谢妍,乜春城,曹佐武.小鼠ZP2mRNA在卵泡颗粒细胞层的表达[J].暨南大学学报,2010,31(2)：116-119.
[64]Sinowatz F, Kolle S, Topfer-Petersen E. Biosynthesis and expression of zona pellucida glycoprotein in mammals[J]. CellsTissues Organs,2001,168(1-2):24-35.
[65]Qing T T, Shi Y, Qin H, et al. Induction of oocyte-like cells from mouse embryonic stem cells byco-culture with ovarian granulosa cells[J]. Differentiation, 2007,75(10):902-911.
[66]Kang Y, Cheng M J, Xu C J. Secretion of estrogen from murine induced pluripotent stem cells co-cultured with ovarian granulosa cells in vitro[J]. Cell Biology International,2011.
[67]Niwa H, Miyazaki J-I, Smith AG Quantitative expression of oct-3/4 defines differentiation dedifferentitation or self-renewal of ES cells[J]. Nat Genet,2000,24(4): 372-376.
[68]Chambers I, Colby D, Robertson M, et al. Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cell[J]. Cell,2003,113(5): 643-655.
[69]Mitsui K, Tokuzawa Y, Itoh H, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[J]. Cell,2003,113(5): 631-642.
[70]Hatano S Y, Tada M, Kimura H. Pluripotential competence of cells associated with Nanog activity[J]. Mechanisms of Development,2005,122(1):67-69.
[71]Constantinescu S. Stemnes, fusion and renewal of hematopoietic and embryonic stem cells[J]. Journal of Cellular and Molecular Medicine,2003,7(2):103-112.
[72]Li Y J, McClintick J, Zhong L, et al. Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor KLF4[J]. Blood,2005,105(2):635-637.
[73]魏素菊,张凯.c-Myc蛋白结构、功能及对肿瘤细胞调控作用[J].河北医药,2009,31(21)：2965-2966.
[74]邓景岳,毕明刚.影响原癌基因c-Myc作用因素的研究进展[J].肿瘤,2009,29(12)：1176-1179.
[2]Owen M E, Cave J, Joyner C J. Clonal analysis in vitro of osteogenic differentiation of marrow CFU-F [J]. J Cell Science,1987,87(5):731-738.
[3]Friedenstein A J, Chailakhyan R K, Gerasimov U V. Bone marrow osteogenic stem cells:in vitro cultivation and transplantation in diffusion chambers [J]. Cell Tissue Kinet,1987,20(3):263-272.
[4]David C C, Reiner C, Carla M et al. Rapid expansion of recycling stem cells in cultrues of plastic-adherent cells from human bone marrow[J]. Proc Natl Acad Sci, 2000,97(7):3213-3218.
[5]Yamaguehi M, Hirayama F, Kanai M,et al. Seru-free eoculture system for ex vivo expansion of human cord blood promotive progenitors and SCID mouse-reconstituting cells using human bone marrow primary stromal cells[J]. Exp Hematol,2001,29(2):174-182.
[6]Pittenger M F, Mackay A M, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells[J]. Science,1999,284(5411):143-147.
[7]Bruder S P, Jaiswal N, Hayneworth S E. Growth kinetics, self-renewal and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation [J]. J Cell Biochem,1997,64(2): 278-285.
[8]Vogel W, Grunerach F, Messam C A, et al. Heterogeneity among human bonemarrow-derived mesenchymal stem cells and neural progenitor cells[J]. Haematologica,2003,88(2):126-133.
[9]MackayAM, Beck S C, Murphy J M, et al. Chondrogentic differentiation of cultured human mesenchymal stem cells from marrow[J]. Tissue Eng,1998,4(4): 415-428.
[10]Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine[J]. MuscleNerve,1995,18(12): 1417-1426.
[11]Wang J S, Shum-Tim D, Galipeau J, et al. Marrow stromal cells for cellular cardiomyoplasty:feasibility and potential clinical advantages [J]. JThorc Cardiovasc Surg,2000,120(5):999-1006.
[12]Sanchez-Ramos J,Song S,Cardozo-Pelaez F,et al. Adult bone marrow s tromal cells differentiate into neural cells in vitro[J]. Exp Neurol,2000,164(2):247-256.
[13]Woodbury D,Schwarz E J, Prockop D J, et al. Adult rat and human bone marrow stromal cells differentiate into neurons[J]. J Neurosci Res,2000,61(4):364-370.
[14]Robert E, Schwarts, Morayma Reyes, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells[J]. J Clin Inves t, 2002,109(10):1291-1302.
[15]Zurita M, Vaquero J. Bone marrow s tromal cells can achieve cure of chronic paraplegic rats:functional and morphological outcome one year after transplantation[J]. Neuroscience Letters,2006,402(10):51-56.
[16]Kopen G C, Prockop D J, Phinney DGMarrow stromalcellsmigrate throughout fore brain and cerebellum, and they dif-ferentiate into astrocytes after injection into neonatalmous brains[J]. Proc NatlAcad SciUSA,1999,96(19):107-111.
[17]Theise N D,Badve S, Saxena R, et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation[J]. Hepatology,2000, 31(1):235-240.
[18]Ferrari G, Angelis GC, Coletta M, et al. Mulscle regeneration by bone marrow-derived myogenic progenitors[J]. Science,1998,279(5356):1528-1530.
[19]Quarto R, Mastrogiacomo M, Cancedda R, et al. Repair of large bone defects with the use of autologous bone marrow stromal cell[J]. New Engl J Med,2001, 344(5):385-386.
[20]Pesce M, Scholer H R. Oct4:control of totipotency and germline determination[J]. Mol Reprod Dev,2000,55(4):452-457.
[21]Niwa H, Miyazaki J-I, Smith A G Quantitative expression of oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells[J]. Nat Genet,2000,24(4): 372-376.
[22]Hart A H, Hartley L, Ibrahim M, et al. Identification, cloning and expression analysis of the pluripotency promoting Nanog genes in mouse and human[J]. Developmental Dynamics,2004,230(1):187-198.
[23]Calaleri F, Scholer H R. Nanog:a new recruit to the embryonic stem cell orchestra[J]. Cell,2003,113(5):551-552.
[24]Wang S H, Tsai M S, Chiang M F, et al. A novel NK typehomeobox gene, ENK (early embryo specific NK), preferentiallyexpressed in embryonic stem cells[J]. Gene Expression Patterns,2003,3(1):99-103.
[25]Pan G J, Pei D Q. Identification of two distinct transactivation domains in the pluripotency sustaining factor nanog[J]. Cell Research,2003,13(6):499-502.
[26]Hart A H, Hartley L, Ibrahim M, et al. Identification, cloning and expression analysis of the pluripotency promoting Nanog genes in mouse and human[J]. Dev Dyn,2004,230(1):187-198.
[27]Liu N, Lu M. The signal transduction pathways and molecules for ES cells self-renewal[J]. Chinese Science Bulletin,2005,50(8):721-726.
[28]Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J]. Cell,2006,126(4): 663-676.
[29]Yet S F, McA'Nulty M M, Folta SC, et al. Human EZF,a Kruppel-like zinc finger protein is expressed in vascular Endothelial cell sandcontain stranscriptional activationand repression domains[J]. J Biol Chem,1998,273(2):1026-1031.
[30]Evans P M, Zhang W, Chen X, et al. Kruppel-like factor 4 is acetylated by p300 and regulates gene transcription via modulation of histone acetylation[J]. J Biol Chem, 2007,282(47):33994-4002.
[31]Cartwright P, McLean C, Sheppard A, et al. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism[J]. Development, 2005,132(5):885-889.
[32]Li Y, McClintick J, Zhong L, et al. Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4[J]. Blood,2005,105(2):635-637.
[33]Lendahl U, Zimmerman L B,Mckay R. CNS stem cells express anew class of intermediate filament protein[J]. Cell,1990,60(4):585-595.
[34]Solway J, Forsythe S M, Halayko A J, et al. Transcriptional regulation of smooth muscle contractile apparatus expression[J]. Am J Respir Crit CareMed,1998,158(5): 100-108.
[35]Debeljak N, Hovrat S, Vouk K, et al. Characterization of the mouse lanosterol 14alpha-demethylase(CYP51), a new member of the evolutionarily most conserved cytoehorme P450 a family[J]. Arch Biochem BioPhys,2000,379(1),37-45.
[36]靳玉甫,覃爱平.卵巢颗粒细胞P450芳香化酶研究进[J].广西医科大学学报,2008,25(6)：972-974.
[37]Sommersberg B, Bulling A, Salzer U, et al. Gap junction communication and connexin 43 gene expression in a rat granulosa cell line:regulation by follicle-stimulating hormone[J]. Biol Reprod,2000,63 (6):1661-1668.
[38]Havelock J C, Rainey W E, Carr B R. Ovarian granulosa cell lines[J]. Mol Cell Endocrinol,2004,228(1-2):67-78.
[39]杨增明,孙青原,夏国良.生殖生物学[M].北京：科学出版社,2005.
[40]卢翠玲,杨巍,胡召元等.颗粒细胞的增殖分化及其在卵泡发育中的作用[J].科学通报,2005,50(21)：2341-2347.
[41]Hunter M H, Sterrett J J. Polycystic ovary syndrome:it's not just infertility [J]. Am Fam Physician,2000,62(5):1079-1090.
[42]Richards J S, Russell D L, Ochsner S, et al. Ovulation:new dimensions and new regulators of the inflammatory-like response[J]. Annu Rev Physiol,2002,64:69-92.
[43]徐晨,轴座民.生殖生物学理论与实践[M].上海：上海科学技术文献出版社,2005.
[44]Zheng W, Magid M S, Kramer E E. Follicle-Stimulating Hormone Receptor Is Expressed in Human Ovarian Surface Epithelium and Fallopian Tube[J]. American Journal of Pathology,1996,148(1):47-53.
[45]Prasad S V, Skinner S M, Carino C, et al. Structure and Function of the Proteins of the Mammalian Zona pellucida, Aachen[J]. Cells Tissues Organs,2000,166(2): 148-164.
[46]张鹏,石玉华,陈子江.生长分化因子-9及生长分化因子9B/骨形态发生蛋白-15与卵巢功能[J].生殖与避孕,2007,27(3)：204-208.
[47]Aaltonen J, Laitinen M P, Vuojolainen K, et al. Human growth differentiation factor 9(GDF-9)and its novel homolog GDF-9B are expressed in oocytes during early folliculogenesis[J]. J Clin Endocrinol Metab,1999,84(8):2744-2750.
[48]Moore R K, Otsuka F, Shimasaki S. Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells[J]. J Biol Chem,2003,278(1):304-310.
[49]Johnson J, Bagley J, Skazni k-Wikiel M, et al. Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood[J]. Cell,2005,122(2):303-315.
[50]贺卫卫,谢琪璇,秦俊文等.骨髓间充质干细胞对损伤卵巢的修复作用[J].暨南大学学报,2010,31(2)：120-124.
[51]Fu X, He Y, Xie C, et al. Bone marrow mesenchymal stem cell transplantation improves ovarian function and structure in rats with chemotherapy-induced ovarian damage[J]. Cytotherapy,2008,10(4):353-363.
[52]Lamoury F M J, Croitoru-lamoury J, Brew B J. Undifferentiated mouse mesenchymal stem cells spontaneously express neural and stem cell markers Oct-4 and Rex-1[J]. Cytotherapy,2006,8(3):228-242.
[53]Tropel P, Noel D, PlatetN, et al. Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow[J]. Exp Cell Res,2004,295(2):395-406.
[54]Hart A H, Hartley L, Ibrahim M, et al. Identification, cloning and expression analysis of the pluripotency promoting nanog genes in mouse and human[J]. Developmental Dynamics,2004,230(1):187-198.
[55]Esposito MT, DiNoto R, Mirabelli P, et al. Culture conditions allow selection of different mesenchymal progenitors from adult mouse bone marrow[J]. Tissue Engineering Part A,2009,15(9):2525-2536.
[56]Woodbury D, Reynolds K, Black I B. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal,and mesodermal genes prior to neurogenesis[J]. J Neurosci Res,2002,96(6):908-917.
[57]Ji K H, Xiong J, Hu K M, et al. Simultaneous expression of Oct4 and genes of three germ layers in single cell-derived multipotent adult progenitor cells[J]. Ann Hematol,2008,87(6):431-438.
[58]Mackenzie S M, McLaughlin E A, Perkins H D, et al. Immunocontraceptive effects on female rabbits infected with recombinant myxoma virus expressing rabbit ZP2 or ZP3[J]. Biol Reprod,2006,74(3):511-21.
[59]Williams Z, Litscher E S, Jovine L, et al. Polypeptide encoded by mouse ZP3 exon-7 is necessary and sufficient for binding of mouse sperm in vitro[J]. J Cell Physiol,2006,207(1):30-39.
[60]Tang J, Xie Q X, Pan S P, et al. Expression of recombinant human zona pellucida-3 protein(rhZP3)in Pichia pastoris[J]. Sheng WuGong Cheng Xue Bao, 2003,19(6):758-762.
[61]Naz RK, Rajesh C. Gene knockouts that cause female infertility:search for novel contraceptive targets[J]. Front Biosci.2005,10:2447-2459.
[62]Mannikko M, Tormala RM, Tuuri T. Association between sequence variations in genes encoding human zona pellucida glycoproteins and fertilization failure in IVF[J].Hum Reprod,2005,20(6):1578-1585.
[63]谢妍,乜春城,曹佐武.小鼠ZP2mRNA在卵泡颗粒细胞层的表达[J].暨南大学学报,2010,31(2)：116-119.
[64]Sinowatz F, Kolle S, Topfer-Petersen E. Biosynthesis and expression of zona pellucida glycoprotein in mammals[J]. CellsTissues Organs,2001,168(1-2):24-35.
[65]Qing T T, Shi Y, Qin H, et al. Induction of oocyte-like cells from mouse embryonic stem cells byco-culture with ovarian granulosa cells[J]. Differentiation, 2007,75(10):902-911.
[66]Kang Y, Cheng M J, Xu C J. Secretion of estrogen from murine induced pluripotent stem cells co-cultured with ovarian granulosa cells in vitro[J]. Cell Biology International,2011.
[67]Niwa H, Miyazaki J-I, Smith AG Quantitative expression of oct-3/4 defines differentiation dedifferentitation or self-renewal of ES cells[J]. Nat Genet,2000,24(4): 372-376.
[68]Chambers I, Colby D, Robertson M, et al. Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cell[J]. Cell,2003,113(5): 643-655.
[69]Mitsui K, Tokuzawa Y, Itoh H, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[J]. Cell,2003,113(5): 631-642.
[70]Hatano S Y, Tada M, Kimura H. Pluripotential competence of cells associated with Nanog activity[J]. Mechanisms of Development,2005,122(1):67-69.
[71]Constantinescu S. Stemnes, fusion and renewal of hematopoietic and embryonic stem cells[J]. Journal of Cellular and Molecular Medicine,2003,7(2):103-112.
[72]Li Y J, McClintick J, Zhong L, et al. Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor KLF4[J]. Blood,2005,105(2):635-637.
[73]魏素菊,张凯.c-Myc蛋白结构、功能及对肿瘤细胞调控作用[J].河北医药,2009,31(21)：2965-2966.
[74]邓景岳,毕明刚.影响原癌基因c-Myc作用因素的研究进展[J].肿瘤,2009,29(12)：1176-1179.