ADAMTS-1在人卵泡颗粒细胞和卵泡液中的表达研究

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;...

Expression of ADAMTS-1 in human follicular granulosa cells and follicular fluid

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.