水牛BMP1基因对颗粒细胞增殖与凋亡的功能研究

doi:10.11843/j.issn.0366-6964.2015.10.009

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (10): 1766-1774.doi: 10.11843/j.issn.0366-6964.2015.10.009

水牛BMP1基因对颗粒细胞增殖与凋亡的功能研究

雷小灿，张海航，崔奎青，刘福航，刘庆友^*，石德顺^*

（广西大学亚热带农业生物资源保护与利用国家重点实验室，南宁 530005）

收稿日期:2015-02-05 出版日期:2015-10-23 发布日期:2015-10-23
通讯作者: 刘庆友，研究员，博士生导师，主要从事动物基因工程研究，E-mail：qyliu2002@126.com；石德顺，研究员，博士生导师，主要从事动物生物技术研究，E-mail：ardsshi@gxu.edu.cn
作者简介:雷小灿（1987-），男，河南辉县人，博士，主要从事家畜卵泡发生的分子机制研究，E-mail：liulincun@163.com *通信作者：刘庆友，研究员，博士生导师，主要从事动物基因工程研究，E-mail：qyliu2002@126.com；石德顺，研究员，博士生导师，主要从事动物生物技术研究，E-mail：ardsshi@gxu.edu.cn
基金资助:
国家转基因重大专项(2011ZX08007-003)；国家高技术研究发展计划(863)项目(2011AA100607)；广西研究生教育创新项目（YCBZ2014001）

Function Analysis of Buffalo BMP1 Gene on Proliferation and Apoptosis of Granulosa Cells

LEI Xiao-can，ZHANG Hai-hang，CUI Kui-qing，LIU Fu-hang，LIU Qing-you^*，SHI De-shun^*

(State Key Laboratory of Conservation and Utilization of Subtropical Agro-bioresources，Guangxi University，Nanning 530005，China)

Received:2015-02-05 Online:2015-10-23 Published:2015-10-23

摘要/Abstract

摘要：

本研究旨在探讨BMP1基因对体外培养水牛颗粒细胞增殖与凋亡的影响，采用qRT-PCR检测体外培养0、24、48、72和96 h水牛颗粒细胞中BMP1基因的表达规律，通过在水牛颗粒细胞培养液中添加BMP1基因重组蛋白和抗BMP1抗体，观察其对体外培养水牛颗粒细胞增殖变化的影响，并应用qRT-PCR和Western blotting方法检测与细胞增殖和细胞凋亡相关因子表达的变化规律。结果显示，随着颗粒细胞体外培养时间的延长，BMP1基因的相对表达量显著上调(P<0.05)，96 h培养组BMP1表达最高。添加BMP1重组蛋白可显著促进水牛颗粒细胞的体外增殖(P<0.05)，并能够显著上调细胞周期关键蛋白Cyclin D1、Cyclin D2和PCNA mRNA表达水平(P<0.05)，同时显著抑制了细胞凋亡信号通路中促凋亡相关因子(Fas、FasL、TNFR1、TNF-α、Cytochrome C、Apaf1、Chop、Caspase-3、Caspase-8和Caspase-9) mRNA表达(P<0.05)。Western blotting检测结果表明，BMP1重组蛋白能够抑制Fas、Bax和Caspase-9蛋白表达，蛋白与mRNA表达一致。而添加抗BMP1抗体的试验则得出相反的结果。综上表明，BMP1基因能通过调控相关基因的表达促进体外培养水牛颗粒细胞的增殖并抑制其凋亡，在水牛卵泡发生过程中具有重要的调控功能，为阐明BMP1基因参与家畜卵泡发生的分子机制奠定基础。

Abstract:

This study focus on the affection of BMP1 gene on proliferation and apoptosis of in vitro cultured buffalo granulosa cells.Expression pattern of BMP1 in different in vitro cultured time of buffalo granulosa cells including 0，24，48，72 and 96 h were determined by qRT-PCR separately.Different concentrations of BMP1 recombinant protein or BMP1 antibody were added to the culture medium，and the effect to proliferation of granulose cells were detected，further effect on expression pattern of cell proliferation and apoptosis associated genes were also analyzed by qRT-PCR and Western blotting.BMP1 gene was significantly increased in the in vitro cultured granulosa cells with the extended recording time(P<0.05)，and reached a peak at 96 h.Adding BMP1 recombinant protein significantly promoted the proliferation of granulosa cells(P<0.05)，as well as up-regulated the expression of Cyclin D1，Cyclin D2 and PCNA genes in mRNA level.In addition，mRNA expression level of pro-apoptosis factors(Fas，FasL，TNFR1， TNF-α，Cytochrome C，Apaf1，Chop，Caspase-3，Caspase-8 and Caspase-9) were down regulated.Furthermore，Western blotting results showed that the protein expression level of Fas，Bax and Caspase-9 was also down-regulated.The contrast results were observed by adding BMP1 antibody investigation.BMP1 promotes proliferation and inhibits apoptosis of in vitro cultured buffalo granulosa cells by regulation the expression pattern of associated genes，which indicated that it took an important role in the follicular development of buffalo，and laid a theoretical foundation to clarify the livestock follicular genesis.

中图分类号:

S823.8⁺3.2

雷小灿，张海航，崔奎青，刘福航，刘庆友，石德顺. 水牛BMP1基因对颗粒细胞增殖与凋亡的功能研究[J]. 畜牧兽医学报, 2015, 46(10): 1766-1774.

LEI Xiao-can，ZHANG Hai-hang，CUI Kui-qing，LIU Fu-hang，LIU Qing-you，SHI De-shun. Function Analysis of Buffalo BMP1 Gene on Proliferation and Apoptosis of Granulosa Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(10): 1766-1774.

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参考文献

［1］CIFTCI H B.Regulation of follicular growth and development in sheep［J］. Iran J Appl Anim Sci，2014，4(1)：21-31.
［2］DUNLOP C E，ANDERSON R A.The regulation and assessment of follicular growth［J］. Scabd J Clin Lab Inv，2014，244(S)：13-17.
［3］SMITH P，WILHELM D，RODGERS R J.Development of mammalian ovary［J］. J Endocrinol，2014，221(3)：R145-R161.
［4］HATZIRODOS N，IRVING-RODGERS H F，HUMMITZSCH K，et al.Transcriptome profiling of granulosa cells of bovine ovarian follicles during growth from small to large antral sizes［J］.BMC Genomics，2014，15(1)：24.
［5］EMORI C，SUGIURA K.Role of oocyte‐derived paracrine factors in follicular development［J］.Anim Sci J，2014，85(6)：627-633.
［6］HOPKINS D R，KELES S，GREENSPAN D S.The bone morphogenetic protein 1/Tolloid-like metalloproteinases［J］.Matrix Biol，2007，26(7)：508-523.
［7］GE G，GREENSPAN D S.Developmental roles of the BMP1/TLD metalloproteinases［J］.Birth Defects Res C Embryo Today，2006，78(1)：47-68.
［8］MARTIÍ E.Expression of chick BMP-1/Tolloid during patterning of the neural tube and somites［J］.Mech Dev， 2000，91(1)：415-419.
［9］FUKAGAWA M，SUZUKI N，HOGAN B L，et al.Embryonic expression of mouse bone morphogenetic protein-1(BMP-1)，which is related to the Drosophila dorsoventral gene tolloid and encodes a putative astacin metalloendopeptidase［J］.Dev Biol，1994，163(1)：175-183.
［10］HÄGGLUND A C，NY A，LEONARDSSON G，et al.Regulation and localization of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the mouse ovary during gonadotropin-induced ovulation 1［J］.Endocrinology，1999，140(9)：4351-4358.
［11］CANTY-LAIRD E，CARRÉ G，MANDON-PÉPIN B，et al.First evidence of bone morphogenetic protein 1 expression and activity in sheep ovarian follicles［J］.Biol Reprod，2010，83(1)：138-146.
［12］BLADER P，RASTEGAR S，FISCHER N，et al.Cleavage of the BMP-4 antagonist chordin by zebrafish tolloid［J］.Science，1997，278(5345)：1937-1940.
［13］JASUJA R，GE G，VOSS N G，et al.Bone morphogenetic protein 1 prodomain specifically binds and regulates signaling by bone morphogenetic proteins 2 and 4［J］.J Biol Chem，2007，282(12)：9053-9062.
［14］WOLFMAN N M，MCPHERRON A C，PAPPANO W N，et al.Activation of latent myostatin by the BMP-1/tolloid family of metalloproteinases［J］.Proc Natl Acad Sci U S A，2003，100(26)：15842-15846.
［15］GE G，HOPKINS D R，HO W，et al.GDF11 forms a bone morphogenetic protein 1-activated latent complex that can modulate nerve growth factor-induced differentiation of PC12 cells［J］.Mol Cell Biol，2005，25(14)：5846-5858.
［16］GE G，GREENSPAN D S.BMP1 controls TGFβ1 activation via cleavage of latent TGF β-binding protein［J］.J Cell Biol，2006，175(1)：111-120.
［17］KIM B，HUANG G，HO W，et al.Bone morphogenetic protein-1 processes insulin-like growth factor-binding protein 3［J］.J Biol Chem，2011，286(33)：29014-29025.
［18］VOS M C，VAN DER WURFF A A，LAST J T，et al.Immunohistochemical expression of MMP-14 and MMP-2，and MMP-2 activity during human ovarian follicular development［J］.Reprod Biol Endocrinol，2014，12(1)：12.
［19］ZHU G，KANG L，WEI Q，et al.Expression and regulation of MMP1，MMP3，and MMP9 in the chicken ovary in response to gonadotropins，sex hormones，and TGFB1［J］.Biol Reprod，2014，90(3)：57.
［20］HERRERA I，CISNEROS J，MALDONADO M，et al.Matrix metalloproteinase(MMP)-1 induces lung alveolar epithelial cell migration and proliferation，protects from apoptosis，and represses mitochondrial oxygen consumption［J］. J Biol Chem，2013，288(36)：25964-25975.
［21］XIAO Q，ZHANG F，GRASSIA G，et al.Matrix metalloproteinase-8 promotes vascular smooth muscle cell proliferation and neointima formation［J］.Aeterioscl Throm Vas，2014，34(1)：90-98.
［22］SHERR C J，ROBERTS J M.CDK inhibitors：positive and negative regulators of G1-phase progression［J］.Gene Dev，1999，13(12)：1501-1512.
［23］BALDIN V，LUKAS J，MARCOTE M J，et al.Cyclin D1 is a nuclear protein required for cell cycle progression in G1.［J］.Gene Dev，1993，7(5)：812-821.
［24］BEUMER T L，ROEPERS-GAJADIEN H L，GADEMAN I S，et al.Involvement of the D-type cyclins in germ cell proliferation and differentiation in the mouse［J］. Biol Reprod，2000，63(6)：1893-1898.
［25］SHIMIZU T，HIRAI Y，MIYAMOTO A.Expression of cyclins and cyclin-dependent kinase inhibitors in granulosa cells from bovine ovary［J］. Reprod Domest Anim，2013，48(5)：e65-e69.
［26］KOZAR K，CIEMERYCH M A，REBEL V I，et al.Mouse development and cell proliferation in the absence of D-cyclins［J］. Cell，2004，118(4)：477-491.
［27］OKTAY K，SCHENKEN R S，NELSON J F.Proliferating cell nuclear antigen marks the initiation of follicular growth in the rat［J］.Biol Reprod，1995，53(2)：295-301.
［28］WANDJI S A，SRSEN V，VOSS A K，et al.Initiation in vitro of growth of bovine primordial follicles.［J］.Biol Reprod，1996，55(5)：942-948.
［29］CHOI Y J，SAEZ B，ANDERS L，et al.D-cyclins repress apoptosis in hematopoietic cells by controlling death receptor Fas and its ligand FasL［J］.Dev Cell，2014，30(3)：255-267.
［30］KWAN J A，SCHULZE C J，WANG W，et al.Matrix metalloproteinase-2(MMP-2) is present in the nucleus of cardiac myocytes and is capable of cleaving poly(ADP-ribose) polymerase(PARP) in vitro［J］.The FASEB J， 2004，18(6)：690-692.
［31］WETZEL M，TIBBITTS J，ROSENBERG G A，et al.Vulnerability of mouse cortical neurons to doxorubicin-induced apoptosis is strain-dependent and is correlated with mRNAs encoding Fas，Fas-Ligand，and metalloproteinases［J］.Apoptosis，2004，9(5)：649-656.
［32］ETHELL D W，KINLOCH R，GREEN D R.Metalloproteinase shedding of Fas ligand regulates β-amyloid neurotoxicity［J］. Curr Biol，2002，12(18)：1595-1600.
［33］BERGERS G，BREKKEN R，MCMAHON G，et al.Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis［J］.Nat Cell Biol，2000，2(10)：737-744.
［34］AHONEN M，POUKKULA M，BAKER A H，et al.Tissue inhibitor of metalloproteinases-3 induces apoptosis in melanoma cells by stabilization of death receptors［J］.Oncogene，2003，22(14)：2121-2134.
［35］TSAO K，TU C，LEE S，et al.Zebrafish scube1(signal peptide-CUB(complement protein C1r/C1s，Uegf，and Bmp1)-EGF(epidermal growth factor) domain-containing protein 1) is involved in primitive hematopoiesis［J］.J Biol Chem，2013，288(7)：5017-5026.

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水牛BMP1基因对颗粒细胞增殖与凋亡的功能研究

Function Analysis of Buffalo BMP1 Gene on Proliferation and Apoptosis of Granulosa Cells

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