【作者】 王欢；

【导师】 潘伯臣；

【作者基本信息】 中国医科大学 ， 妇产科学， 2010， 硕士

【摘要】 目的肝素结合性表皮生长因子(heparin-binding epidermal growth factor-like growth factor,HB-EGF)是表皮生长因子家族的新成员。在细胞中,首先合成的是其前体,镶嵌于细胞膜上,故称膜结合型HB-EGF (proHB-EGF)。之后,在特异性去整合素-金属蛋白酶(a disintegrin and metalloprotease, ADAM)的作用下,proHB-EGF的细胞外域(ectodomain)被切下,脱落成为可溶性的成熟体,即溶解型HB-EGF (sHB-EGF)。sHB-EGF是多种细胞的分裂原,促进细胞增殖。proHB-EGF也具有生物活性,主要为抑制细胞生长、诱导凋亡。ADAM蛋白是一类锚定于细胞膜的细胞表面蛋白家族,ADAM蛋白可能在细胞外基质的水解、细胞-细胞、细胞-基质的粘连、细胞融合及信号转导等细胞活动中具有重要作用,广泛参与各种正常的生理活动。在人体,有21种ADAM亚型被发现,它们的表达存在显著的组织特异性：其中ADAM2、7、18、19、20、21、30在睾丸特异表达,ADAM8、28在造血细胞特异表达,本实验在剩余的ADAM亚型(ADAM9、10、11、12、15、17、19、22、23、32、33、ADAMDEC1)中筛选黄体发育和消退过程中特异性水解膜结合型HB-EGF的ADAM亚型,为人工干预黄体的发育与消退提供新的靶点。方法取行体外受精-胚胎移植患者的黄素化颗粒细胞,提取颗粒细胞RNA,用Primer5.0软件设计各亚型的引物,再用RT-PCR的方法筛选ADAM亚型在颗粒细胞中的表达情况,并通过测序方法进一步验证。结果RT-PCR方法检测证实ADAM9和ADAM17在卵巢颗粒细胞中有表达,并且通过测序得到了进一步的证实。结论卵巢颗粒细胞中ADAM基因的表达亚型为ADAM9和ADAM17,推测ADAM9和／或ADAM17可能为黄体发育和消退过程中特异性水解膜结合型HB-EGF的ADAM亚型。实验结果为寻找人工干预黄体发育与消退的新靶点提供了有益的线索。更多还原

【Abstract】 ObjectiveHB-EGF is a new member of epidermal growth factor families. In the cell, the first synthesis of its precursor (proHB-EGF), embedded in the membrane, so called membrane-bound HB-EGF. Later, in the function of specific disintegrin-metalloproteinase (a disintegrin and metalloprotease, ADAM), proHB-EGF extracellular domain (ectodomain) has been cut off, falling into soluble mature body, that is solubilty HB-EGF (sHB-EGF). sHB-EGF is a multi-cell mitogen, promoting cell proliferation. proHB-EGF also have biological activity, mainly inhibit cell growth and induce apoptosis. ADAM is a type discovered in recent years anchored in the cell membrane of the cell-surface protein family, ADAM proteins may be the hydrolysis of extracellular matrix, cell-cell, cell-matrix adhesion, cell fusion and signal transduction and other cell activity plays an important role,which is broad participate in a variety of normal physiological activity. In the human body, there are 21 kinds of ADAM types were found, whose expression has tissue-specific:ADAM2, 7,18,19,20,21,30 in the testis-specific expression, ADAM8,28 in the haematopoietic expression, this experiment in the remaining subtypes of ADAM Screening corpus luteum during development and fade-specific hydrolysis of membrane-bound HB-EGF in disintegrin-metalloproteinase (a disintegrin and metalloprotease, ADAM) subtypes, in order to providing a new hegemony of the corpus luteum of the development and the fade of human intervention.MethodRecipe the patients’luteinized granulosa cells who are in vitro fertilization-embryo transfer, extract the RNA of granule cells, using Primer 5.0 software design the primers of ADAM subtypes, and then using RT-PCR method to screen ADAM subtypes in granulosa cells and to further verified by sequencing. ResultRT-PCR confirmed that ADAM9 and ADAM 17 were detected in ovarian granulosa cells to express and by sequencing has been further confirmed.ConclusionThe expression of ADAM gene subtypes in ovarian granulosa cells is ADAM9 and ADAM 17, speculated that ADAM9 and/or ADAM 17 may be the subtypes that is specific hydrolysis membrane-bound HB-EGF in the development and extinction of corepus luteum, so as to provide a new treatment clues of the development of corpus luteum and the fade of human intervention.更多还原