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目的探讨Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶(ADAMTS-1)mRNA和蛋白在人卵泡壁颗粒细胞(GCs)和卵丘颗粒细胞(CCs)中的表达水平及变化。方法收集2014年3月至2016年6月期间在华中科技大学同济医学院生殖医学中心接受ICSI治疗的患者卵泡液,其中大卵泡(直径≥18 mm)和小卵泡(≤10 mm)各86份,离心后上清用于Western Blot和ELISA实验,沉淀用密度梯度离心法分离纯化得到GCs;收集脱颗粒细胞的培养液直接离心法获得CCs。GCs和CCs均分别提取mRNA和蛋白,荧光定量PCR检测GCs和CCs中ADAMTS-1 mRNA水平,Western Blot检测ADAMTS-1蛋白水平,比较ADAMTS-1 mRNA和蛋白在不同颗粒细胞中的表达差异。结果 ADAMTS-1 mRNA在大卵泡GCs、小卵泡GCs、CCs中均有表达,且其在小卵泡GCs中表达量最高,显著高于CCs(P<0.05)。ADAMTS-1蛋白在大卵泡GCs、小卵泡GCs、CCs及卵泡液中均有表达,且存在成熟ADAMTS-1蛋白(85 KDa)及前体(110 KDa)两种形式;其在CCs中的表达量最高,显著高于大、小卵泡的GCs(P<0.05),在小卵泡液中的含量显著高于大卵泡液(P<0.01)。结论 ADAMTS-1 mRNA在人卵泡GCs和CCs中均有表达,ADAMTS-1蛋白在人卵泡GCs、CCs和卵泡液中均存在前体和成熟形式,且以成熟形式为主。ADAMTS-1在人卵泡GCs、CCs和卵泡液中的表达特点及变化可能与其在卵泡发育过程中的功能相关。
Abstract：Objective:To explore the expression levels of ADAMTS-1 mRNA and protein in granulosa cells(GCs) and cumulus cells(CCs) of follicles and follicular fluid.Methods:The samples of follicular fluid of ICSI-treated patients were collected at the Reproductive Medicine Center of Tongji Medical College of Huazhong University of Science and Technology from March 2014 to June 2016,including 86 large follicles(diameter ≥18 mm) and 86 small follicles(≤10 mm).The samples of follicular fluid were centrifuged and the supernatants were used for Western blot and ELISA,respectively.GCs were separated by using density gradient centrifugation,and CCs were obtained by directly centrifuging the culture solution of degranulosa cells.The mRNA and proteins of ADAMTS-1 in GCs and CCs were determined by qRT-PCR and Western blot analysis.The relative expression levels of ADAMTS-1 mRNA and protein were compared in different cells.Results:ADAMTS-1 mRNA was expressed in both of GCs and CCs of large follicles and small follicles,and its expression was the highest in GCs of small follicles,which was significantly higher than that of CCs(P<0.05).ADAMTS-1 protein was expressed in GCs,CCs and follicular fluid of large and small follicles.There are two forms of maturity ADAMTS-1 protein(85 KDa) and precursor(110 KDa). ADAMTS-1 protein expression level in CCs was significantly higher than that of GCs from large or small follicles(P<0.05),and its content in follicular fluid of small follicles was significantly higher than that of large follicles(P<0.01).Conclusions:ADAMTS-1 mRNA is expressed both in GCs and CCs.ADAMTS-1 protein in GCs,CCs and follicular fluid has the precursor and maturity form,and mainly in maturity form.The expression characteristics and changes of ADAMTS-1 in GCs,CCs and follicular fluid may be related to its function during follicular development.
[1] Reymond A,Marigo V,Yaylaoglu MB,et al.Human chromosome 21 gene expression atlas in the mouse[J].Nature,2002,420:582-586.
[2] Yunq Y,Maman E,Konopnicki S,et al.ADAMTS-1,new human ovulatory gene and a cumulus marker for fertilization capacity[J].Mol Cell Endocrinol,2010,328:104-108.
[3] 蔡倩方,艾继辉,章汉旺.ADAMTS-1在排卵过程中的作用与调控[J].中国优生与遗传杂志,2008,16:17-19.
[4] Yu L,Mei C,Xue Z,et al.Bone morphogenetic protein 6 expression in cumulus cells is negatively associated with oocyte maturation[J].Hum Fertil (Camb),2019,9:1-8.
[5] Zhang L,Liang Y,Liu Y,et al.The role of brain-derived neurotrophic factor in mouse oocyte maturation in vitro involves activation of protein kinase B[J].Theriogenology,2010,73:1096-1103.
[6] Shindo T,Kurihara H,Kuno K,et al.ADAMTS-1:a metalloproteinase-disintegrin essential for normal growth,fertility,and organ morphology and function[J].J Clin Invest,2000,105:1345-1352.
[7] 申锷,陈瑞珍,杨英珍,等.ADAMTS-1的基础研究及临床意义[J].国际病理科学与临床杂志,2006,26:29-30.
[8] Castro AF,Rebhun JF,Clark GJ,et al.Rheb binds tuberous sclerosis complex 2 (TSC2) and promotes S6 kinase activation in a rapamycin-and farnesylation-dependent manner[J].J Biol Chem,2003,278:32493-32496.
[9] Shozu M,Minami N,Yokoyama H,et al.ADAMTS-1 is involved in normal follicular development,ovulatory process and organization of the medullary vascular network in the ovary[J].J Mol Endocrinol,2005,35:343-355.
[10] Espey LL,Yoshioka S,Russell DL,et al.Ovarian expression of a disintegrin and metalloproteinase with thrombospondin motifs during ovulation in the gonadotropin-primed immature rat[J].Biol Reprod,2000,62:1090-1095.
[11] Russell DL,Dovle KMH,Ochsner SA,et al.Processing and localization of ADAMTS-1 and proteolytic cleavage of versican during cumulus matrix expansion and ovulation[J].J Biol Chem,2003,278:42330-42339.
[12] Brown HM,Dunning KR,Robker RL,et al.ADAMTS-1 cleavage of versican mediates essential structural remodeling of the ovarian follicle and cumulus-oocyte matrix during ovulation in mice[J].Biol Reprod,2010,83:549-557.
[13] Dunning KR,Lane M,Brown HM,et al.Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes[J].Hum Reprod,2007,22:2842-2850.
[14] Cookingham LM,Van Voorhis BJ,Ascoli M.Do alterations in follicular fluid proteases contribute to human infertility?[J].J Assist Reprod Genet,2015,32:737-745.
[15] Brown HM,Dunning KR,Robker RL,et al.Requirement for ADAMTS-1 in extracellular matrix remodeling during ovarian folliculogenesis and lymphangiogenesis[J].Dev Biol,2006,300:699-709.
基本信息:
中图分类号:R714.8
引用信息:
[1]陈梅,张玲.ADAMTS-1在人卵泡颗粒细胞和卵泡液中的表达研究[J].生殖医学杂志,2020,29(06):798-802.
基金信息:
国家自然科学基金青年基金(81000274)
2020-06-15
2020-06-15