脑源性神经营养因子及其受体在人卵巢的表达与卵泡发育的关系

Expression of brain-derived neurotrophin factor and its receptor tyrosine kinase receptor B in human ovary and their significance

目的 :研究脑源性神经营养因子 (BDNF)及其受体———酪氨酸蛋白激酶受体B(TrkB)在人卵巢中的表达。初步探讨其与卵泡发育的关系。方法 :取排卵前期人卵巢组织和来自体外受精 胚胎移植 (IVF ET)周期的取卵日卵泡液、人卵巢黄素化壁层颗粒细胞 (hMGLCs)、人卵巢黄素化卵丘颗粒细胞 (hCGLCs)、未受精卵母细胞或未成熟卵母细胞。用免疫组织化学SABC法研究BDNF在人卵巢组织中不同发育阶段卵泡的表达 ,酶联免疫吸附实验 (ELISA)方法检测BDNF在人卵泡液 ,hMGLCs和hCGLCs无血清培养上清液中的表达。用免疫组织化学SABC法研究TrkB在人卵巢组织不同发育阶段卵泡的表达 ,免疫细胞化学SABC法研究TrkB在hMGLCs,hCGLCs和未受精或未成熟卵母细胞的表达 ,Westernblotting方法检测TrkB在hMGLCs和hCGLCs的表达及其差异。结果 :人卵泡液中存在BDNF ,BDNF表达于初级卵泡和次级卵泡的颗粒细胞及窦状卵泡的卵丘颗粒细胞。TrkB表达于初级卵泡和次级卵泡的颗粒细胞、窦状细胞的卵丘颗粒细胞、壁层颗粒细胞及卵母细胞 ,壁层颗粒细胞表达量高于卵丘颗粒细胞。结论 :在人卵巢BDNF及其受体TrkB可能 (通过旁分泌和自分泌的方式 )参与颗粒细胞功能的完成和卵母细胞发育的调控。

Objective:To study the expressions of brain derived neurotrophin factor (BDNF) and its receptor tyrosine kinase receptor B (TrkB) in human ovary and evaluate the relationship between the expressions and the development of follicle.Methods:Human ovaries were retrieved before ovulation from the patients who need ovary resection.Follicular fluid,human mural granulosa luteal cells (hMGLCs),human cumulus granulosa luteal cells (hCGLCs) and unfertilized oocytes or immutured oocytes were obtained from women who undergoing soper ovulation induction for in vitro fertilization embryo transfer (IVF ET).After a 24 h initial culture,hMGLCs and hCGLCs were cultured in serum free 199 medium.SABC immunohistochemistry was used to study the expressions of BDNF and TrkB in the different follicle phase.ELISA was used to examine the expression of BDNF in human follicular fluid,culture medium of hMGLCs and hCGLCs.SABC immunocytochemistry was used to study the expression of TrkB in hMGLCs,hCGLCs and unfertilized oocytes or immutured oocytes.Western blotting was used to analyse the expression of TrkB in hMGLCs and hCGLCs.Results:These experiments demonstrated for the first time the presence of BDNF in the serum free medium 199 of hCGLCs culture and human granulosa cells cytoplasm of primary follicle and secondary follicle.BDNF also exists in follicular fluid also.It was for the first time that TrkB was identied to present not only on the human granulosa cells membrance of primary follicle and secondary follicle but also on the membrance of human mural granulosa cells,cumulus granulosa cells,oocytes of antral follicle.Expression of TrkB in hMGLCs was higher than hCGLCs.Conclusion:It is possible that BDNF and TrkB may effect on regulation and modulation of steroidogenesis of granulosa cells and maturation of oocytes (through paracrine and autocrine action). [