脑源性神经营养因子抑制牛卵泡颗粒细胞凋亡
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摘要
为研究脑源性神经营养因子(Brain-derived neurotrophic factor,BDNF)对牛卵泡颗粒细胞凋亡的影响。首先利用CCK8检测了不同浓度(0,10,50,100,200 ng/m L)的BDNF处理牛卵泡颗粒细胞24 h之后细胞活性变化情况,发现50,100,200 ng/m L的BDNF都能够极显著的增加颗粒细胞的活性(P<0. 01)。以100 ng/m L作为BDNF最适处理浓度,利用流式细胞仪,通过线粒体膜电位检测(JC-1)发现,BDNF组的红绿荧光相对比例极显著的升高(P<0. 01)。接着利用实时荧光定量PCR方法,发现BDNF导致P53、Bax和Caspase-3的mRNA表达水平极显著降低(P<0. 01),而Bcl-2无显著变化(P>0. 05)。最后通过siRNA干扰内源性的BDNF的合成(P<0. 01),利用实时荧光定量PCR方法发现,干扰BDNF之后,P53极显著的升高(P<0. 01),Bax显著升高(P<0. 05),Bcl-2和Caspase-3无显著差异(P>0. 05)。结果表明:BDNF对牛卵泡颗粒细胞的凋亡有抑制作用,为牛卵泡的发育和颗粒细胞功能的研究提供了新的途径。
The purpose of this study was to investigate the effects of brain-derived neurotrophic factor on apoptosis of bovine granulosa cells. To examine whether BDNF affects apoptosis of bovine granulosa cells,cell viability was examined by cell counting kit-8( CCK-8) assay at 24 h after treatment with BDNF at various concentrations( 0,10,50,100 and 200 ng/m L). The results showed that BDNF increased significantly the viability of bovine granulosa cells after treatment with BDNF at 50,100 or 200 ng/m L at 24 h( P<0. 01). In order to further determine whether BDNF would inhibit the apoptosis of bovine granulosa cells,mitochondrial membrane potential( sensitive fluorescent probe JC-1) was examined by flow cytometry analysis at 24 h after treatment with BDNF( 100 ng/m L).The results showed that BDNF increased significantly the ratio of red/green fluorescent density( P<0. 01). Real-time quantitative PCR was used to examine the levels of mRNA about apoptosis. The results showed that BDNF reduced significantly the expression levels of mRNA for P53,Bax and Caspase-3( P<0. 01),while the expression levels of mRNA for Bcl-2 was not significantly different( P>0. 05). The mRNA expression levels of genes related to apoptosis were examined after bovine granulosa cells were transfected with si BDNF. The results showed that the mRNA expression levels of P53( P<0. 01) and Bax( P<0. 05) were increased significantly,while the expression levels of mRNA for Bcl-2 and Caspase-3 were not significantly different( P>0. 05). These results suggested that BDNF inhibited the apoptosis of bovine granulosa cells. This study provided a novel orientation for underlying function of BDNF in granulosa cells and follicular development.
The purpose of this study was to investigate the effects of brain-derived neurotrophic factor on apoptosis of bovine granulosa cells. To examine whether BDNF affects apoptosis of bovine granulosa cells,cell viability was examined by cell counting kit-8( CCK-8) assay at 24 h after treatment with BDNF at various concentrations( 0,10,50,100 and 200 ng/m L). The results showed that BDNF increased significantly the viability of bovine granulosa cells after treatment with BDNF at 50,100 or 200 ng/m L at 24 h( P<0. 01). In order to further determine whether BDNF would inhibit the apoptosis of bovine granulosa cells,mitochondrial membrane potential( sensitive fluorescent probe JC-1) was examined by flow cytometry analysis at 24 h after treatment with BDNF( 100 ng/m L).The results showed that BDNF increased significantly the ratio of red/green fluorescent density( P<0. 01). Real-time quantitative PCR was used to examine the levels of mRNA about apoptosis. The results showed that BDNF reduced significantly the expression levels of mRNA for P53,Bax and Caspase-3( P<0. 01),while the expression levels of mRNA for Bcl-2 was not significantly different( P>0. 05). The mRNA expression levels of genes related to apoptosis were examined after bovine granulosa cells were transfected with si BDNF. The results showed that the mRNA expression levels of P53( P<0. 01) and Bax( P<0. 05) were increased significantly,while the expression levels of mRNA for Bcl-2 and Caspase-3 were not significantly different( P>0. 05). These results suggested that BDNF inhibited the apoptosis of bovine granulosa cells. This study provided a novel orientation for underlying function of BDNF in granulosa cells and follicular development.
引文
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[2]尤劲松,胡随瑜.卵泡闭锁与颗粒细胞凋亡[J].临床与病理杂志,2000(6):487-490.
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[4] Paredes A,Romero C,Dissen G A,et al. TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary[J]. Developmental Biology,2004,267(2):430-449.
[5] Dissen G A,Hirshfield A N,Malamed S,et al. Expression of neurotrophins and their receptors in the mammalian ovary is developmentally regulated:changes at the time of folliculogenesis[J]. Endocrinology,1995,136:4681-4692.
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[8] B S D,Bo F,M S R. Immunocytochemical evidence for the presence and location of the neurotrophin-Trk receptor family in adult human preovulatory ovarian follicles[J]. American Journal of Obstetrics&Gynecology,2006,194(4):1129-1134.
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[12]李纯锦.牛和人精子中脑源性神经营养因子和神经生长因子的表达及功能研究[D].长春:吉林大学,2010.
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[14]于佳鑫,蒋彦文,陈树雄,等.雌激素对小鼠子宫BDNF、NGF表达的影响[J].吉林农业大学学报,2016,38(2):198-202.
[15] Sadeu J C,Doedée A M C M,Neal M S,et al. Neurotrophins(BDNF and NGF)in follicular fluid of women with different infertility diagnoses[J]. Reproductive Biomedicine,2012,24(2):174-179.
[16] Dorfman M D,Kerr B,Garciarudaz C,et al. Neurotrophins acting via TRKB receptors activate the JAGGED1-NOTCH2cell-cell communication pathway to facilitate early ovarian development[J]. Endocrinology,2011,152(12):5005.
[17] Chun S Y,Hsueh A J. Paracrine mechanisms of ovarian follicle apoptosis[J]. Journal of Reproductive Immunology,1998,39(1-2):63-75.
[18]张婷.颗粒细胞存活或凋亡因子对卵泡生长与闭锁的调控[J].科技信息,2013(5):212.
[19]李洁云.热应激对小鼠卵巢卵泡闭锁和颗粒细胞凋亡的影响及其机理研究[D].北京:中国农业大学,2016.
[20] Peng J Y,An X P,Fang F,et al. MicroRNA-10b suppresses goat granulosa cell proliferation by targeting brain-derived neurotropic factor[J]. Domestic Animal Endocrinology,2015,54:60.
[21] Xie M,Li M,Zhou J,et al. Brain-derived neurotrophic factor promotes human granulosa-like tumor cell steroidogenesis and proliferation by activating the FSH receptor-mediated signaling pathway[J]. Scientific Reports,2017,7(1):180.
[22] Li C,Chen C,Chen L,et al. BDNF-induced expansion of cumulus-oocyte complexes in pigs was mediated by microRNA-205[J]. Theriogenology,2016,85(8):1357-1506.
[23] Sun Yongfeng,Sun Yanling,Chen Lu,et al. Expression of nurotrophin 4 and it's receptor Tyrosine Kinase B in reproductive tissues during the follicular and luteal phases in cows[J].J Anim Sci,2011,3(24):336-343.
[24] Hetz C,Vitte P,Bombrun A,et al. Bax channel inhibitors prevent mitochondrion-mediated apoptosis and protect neurons in a model of global brain ischemia[J]. Journal of Biological Chemistry,2005,280(52):42960-42970.
[25] Grubb B J. Developmental biology,eighth edition[J]. Yale Journal of Biology&Medicine,2007,80(2):92-93.
[26] Aharoni-Simon M,Shumiatcher R,Yeung A,et al. Bcl-2regulates reactive oxygen species signaling and a redox-sensitive mitochondrial proton leak in mouse pancreaticβ-cells[J]. Endocrinology,2016,157(6):en20151964.
[27] Gross A,Jockel J,Wei M C,et al. Enforced dimerization of BAX results in its translocation,mitochondrial dysfunction and apoptosis[J]. Embo Journal,2014,17(14):3878-3885.
[28] Hsu Y T,Wolter K G,Youle R J. Cytosol-to-membrane redistribution of Bax and Bcl-XL during Apoptosis[J]. Proceedings of the National Academy of Sciences of the United States of America,1997,94(8):3668-3672.
[29] Salvesen G S. Caspases:opening the boxes and interpreting the arrows[J]. Cell Death&Differentiation,2002,9(1):3-5.
[30] Chen J Z,Ji C N,Gu S H,et al. Over-expression of Bimα3,a novel isoform of human Bim,result in cell apoptosis[J]. International Journal of Biochemistry&Cell Biology,2004,36(8):1554-1561.
[31] Perez G I,Robles R,Knudson C M,et al. Prolongation of ovarian lifespan into advanced chronological age by Bax-deficiency[J]. Nature Genetics,1999,21(2):200-203.
[2]尤劲松,胡随瑜.卵泡闭锁与颗粒细胞凋亡[J].临床与病理杂志,2000(6):487-490.
[3] Yuan W,Giudice L C. Programmed cell death in human ovary is a function of follicle and corpus luteum status[J]. The Journal of Clinical Endocrinology and Metabolism,1997,82(9):3148-3155.
[4] Paredes A,Romero C,Dissen G A,et al. TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary[J]. Developmental Biology,2004,267(2):430-449.
[5] Dissen G A,Hirshfield A N,Malamed S,et al. Expression of neurotrophins and their receptors in the mammalian ovary is developmentally regulated:changes at the time of folliculogenesis[J]. Endocrinology,1995,136:4681-4692.
[6] Yi K L,Zhou X,Shi D S,et al. The mRNA expression of brain-derived neurotrophic factor in oocytes and embryos and its effects on the development of early embryos in cattle[J].Animal,2008,2(12):1786-1794.
[7]芭帕婉,侯晓峰,王春强,等.脑源性神经营养因子在猪卵泡中的表达[J].吉林农业大学学报,2012,34(6):677-681.
[8] B S D,Bo F,M S R. Immunocytochemical evidence for the presence and location of the neurotrophin-Trk receptor family in adult human preovulatory ovarian follicles[J]. American Journal of Obstetrics&Gynecology,2006,194(4):1129-1134.
[9] Mirshokraei P,Hassanpour H,Rahnama A,et al. Gene expression of BDNF and its receptors,TrkB and p75 in the uterus and oviduct of pregnant and non-pregnant ewes[J]. Research in Veterinary Science,2013,95(1):164-168.
[10]陈璐.脑源性神经营养因子及神经营养因子4对大鼠促性腺激素分泌及其受体表达的影响[D].长春:吉林大学,2012.
[11] Childs A J,Bayne R A L,Murray A A,et al. Differential expression and regulation by activin of the neurotrophins BDNF and NT4 during human and mouse ovarian development[J].Developmental Dynamics,2010,239(4):1211-1219.
[12]李纯锦.牛和人精子中脑源性神经营养因子和神经生长因子的表达及功能研究[D].长春:吉林大学,2010.
[13] Boyd J G,Gordon T. Glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor sustain the axonal regeneration of chronically axotomized motoneurons in vivo[J].Experimental Neurology,2003,183(2):610-61`9.
[14]于佳鑫,蒋彦文,陈树雄,等.雌激素对小鼠子宫BDNF、NGF表达的影响[J].吉林农业大学学报,2016,38(2):198-202.
[15] Sadeu J C,Doedée A M C M,Neal M S,et al. Neurotrophins(BDNF and NGF)in follicular fluid of women with different infertility diagnoses[J]. Reproductive Biomedicine,2012,24(2):174-179.
[16] Dorfman M D,Kerr B,Garciarudaz C,et al. Neurotrophins acting via TRKB receptors activate the JAGGED1-NOTCH2cell-cell communication pathway to facilitate early ovarian development[J]. Endocrinology,2011,152(12):5005.
[17] Chun S Y,Hsueh A J. Paracrine mechanisms of ovarian follicle apoptosis[J]. Journal of Reproductive Immunology,1998,39(1-2):63-75.
[18]张婷.颗粒细胞存活或凋亡因子对卵泡生长与闭锁的调控[J].科技信息,2013(5):212.
[19]李洁云.热应激对小鼠卵巢卵泡闭锁和颗粒细胞凋亡的影响及其机理研究[D].北京:中国农业大学,2016.
[20] Peng J Y,An X P,Fang F,et al. MicroRNA-10b suppresses goat granulosa cell proliferation by targeting brain-derived neurotropic factor[J]. Domestic Animal Endocrinology,2015,54:60.
[21] Xie M,Li M,Zhou J,et al. Brain-derived neurotrophic factor promotes human granulosa-like tumor cell steroidogenesis and proliferation by activating the FSH receptor-mediated signaling pathway[J]. Scientific Reports,2017,7(1):180.
[22] Li C,Chen C,Chen L,et al. BDNF-induced expansion of cumulus-oocyte complexes in pigs was mediated by microRNA-205[J]. Theriogenology,2016,85(8):1357-1506.
[23] Sun Yongfeng,Sun Yanling,Chen Lu,et al. Expression of nurotrophin 4 and it's receptor Tyrosine Kinase B in reproductive tissues during the follicular and luteal phases in cows[J].J Anim Sci,2011,3(24):336-343.
[24] Hetz C,Vitte P,Bombrun A,et al. Bax channel inhibitors prevent mitochondrion-mediated apoptosis and protect neurons in a model of global brain ischemia[J]. Journal of Biological Chemistry,2005,280(52):42960-42970.
[25] Grubb B J. Developmental biology,eighth edition[J]. Yale Journal of Biology&Medicine,2007,80(2):92-93.
[26] Aharoni-Simon M,Shumiatcher R,Yeung A,et al. Bcl-2regulates reactive oxygen species signaling and a redox-sensitive mitochondrial proton leak in mouse pancreaticβ-cells[J]. Endocrinology,2016,157(6):en20151964.
[27] Gross A,Jockel J,Wei M C,et al. Enforced dimerization of BAX results in its translocation,mitochondrial dysfunction and apoptosis[J]. Embo Journal,2014,17(14):3878-3885.
[28] Hsu Y T,Wolter K G,Youle R J. Cytosol-to-membrane redistribution of Bax and Bcl-XL during Apoptosis[J]. Proceedings of the National Academy of Sciences of the United States of America,1997,94(8):3668-3672.
[29] Salvesen G S. Caspases:opening the boxes and interpreting the arrows[J]. Cell Death&Differentiation,2002,9(1):3-5.
[30] Chen J Z,Ji C N,Gu S H,et al. Over-expression of Bimα3,a novel isoform of human Bim,result in cell apoptosis[J]. International Journal of Biochemistry&Cell Biology,2004,36(8):1554-1561.
[31] Perez G I,Robles R,Knudson C M,et al. Prolongation of ovarian lifespan into advanced chronological age by Bax-deficiency[J]. Nature Genetics,1999,21(2):200-203.