The Research On The Effects Of Glial Cell Line-derived Neurotrophic Factor On Proliferation

Glial cell derived neurotrophic factor (glial cell line-derived neurotrophic factor, GDNF) were found in 1993, Lin, and so on, a new active neurotrophic factor, in recent years, let the reproductive scholars interested in GDNF can not only weeks out organs in expression, and there is evidence that the expression quantity higher than that of the central nervous system, which is based on the expression level by for ovarian.Expression is also very significant in testicular tissue.Ovary connected to the nerves and blood vessels not only, also connected with follicle, at the same time can find ectopic transplantation of ovarian tissue quickly grow nerve fibers.GDNF in ovarian tissue increased, and the distribution and adult ovaries in urinary system and throughout the entire embryo development stage, mainly concentrated in follicular granulosa cells.GDNF mRNA expression level in mice with follicular development gradually become significant, until the peak before ovulation.Based on the above situation, GDNF gene protein in mice mainly distributed in the egg and granulosa cells and stromal cells.Kawamura et al. conducted a DNA microarray analysis of the experiment, also confirmed DGNF’s important role in the mice, the subsequent experiment results show that GDNF widely expressed in ovary, mice in the egg and high cell, granular cell and cell membranes are expressed in the cell.From newborn mice to adult mice have GDNF mRNA expression in ovarian, throughout the development stage, mainly in the follicle mesenchymal cells of the egg, egg in sinus follicle high cell, granular cell and cell membrane cells were also expressed.In vitro maturation of oocytes from experience training women (IVM) of oocytes experimental observation found that in the last stage of the follicle stimulating the expression of GDNF protein in the ovaries is to expand the scope of, mainly distributed in granulosa cells were exciting.Gradually, through the obtained data matching and various kinds of mammals, we can come to the conclusion that the expression of GDNF and its receptor model, in the adult female/female ovaries GDNF in paracrine and autocrine manner affect oocyte and egg high cells.Although the GDNF in reproductive research has been more, but people ovarian research is still little, there is evidence that people luf particles within the nucleus and cytoplasm GDNF, GFRa-1, the expression of Ret, and the FHS and hCG can increase the expression of GDNF in granulosa cells, and promote mature follicle in a paracrine manner. In the process of ovarian tissue in vitro culture, join GDNF can promote follicle growth and maturity, but for GDNF in granulosa cells and oocytes respective mechanism?This study KGN granulosa cells and human oocytes as the research object, the detection of GDNF and the expression of receptors in human oocytes, observation of GDNF stimulus for KGN granulosa cells in vitro culture play a role, and discussed its role in the mechanism.ObjectiveTo evaluate whether GDNF promotes the in vitro development of human preantral follicles and Study the effects of GDNF in the process of KGN cell proliferation, and GDNF after stimulation KGN cell gene expression changes, provide beneficial theoretical and experimental basis for clinical application.Methods1.Collect ovarian cyst surgery in patients with normal ovarian cortex, adopt the method of vitrification freezing line processing frozen, thawed after digestive enzyme digestion method, after digestion mechanical method in the microscopic primordium follicle and primary follicles, in FA gel, GDNF stimulation, selecting the appropriate concentration, observation of GDNF after stimulation in vitro cultivation of follicle changes.2.Culture KGN cells in vitro, extend into 96 hole dish, GDNF stimulus given different concentration respectively, observing 24 h,48 h after CCK 8 stimulating cell proliferation.3. Cultivate KGN cells in vitro, in 6 hole dish after the wedding, treatment group given 5 ng/ml GDNF stimulation,24 h,48 h after collecting experimental group and control group respectively 1 ml of liquids, detection of estrogen and progesterone changes between the 2 groups.4. Cultivate KGN cells in vitro,24 h after hole in 6 batches of plating, processing line hunger after 6 h, give 1 ul containing GDNF reagent to the test group GDNF concentration for 5 ul/ml,6 h after cracking cells and extract the RNA, do the PCR detection of genetic changes.5. Cultivate KGN cells in vitro, to extend into the 12 hole dish, line up processing, using immunohistochemistry and RT-PCR method to detect its receptor family of GDNF receptor tyrosine kinase receptor RET (rearranged during transfeetion) expression.Result1. Enzyme+mechanical joint separation follicle after put in FA gel, given different concentrations of GDNF stimulation, with the increase of training days, each follicle diameter and secretion of E2 levels were significantly increased (P< 0.05); And in vitro culture 2,4,6, and 8 days,10 ng/mlGDNF group and 100 ng/mlGDNF follicle diameter and secretion E2 level in growth is significantly higher than control group and 1 ng/mlGDNF group (P< 0.05).2. The different levels of GDNF affect different influence on the growth of granular cell line:high concentration of GDNF inhibition of cell proliferation, low concentration of GDNF promote cell proliferation.Among them 100 ng/ml significantly inhibit than control group (P< 0.05),5 ng/ml group than the control group significantly promotes the proliferation (P< 0.05).3.5 ng/KGN mlGDNF cells after no significant influence on granulosa cells of female progestational hormone (P> 0.05).4. FSH and its receptor mRNA expression has changed after 5 ng/mlGDNF stimulation, expressing quantity increased (P<0.05).5. Receptor protein of GDNF RET expression in granulosa cells, located in the cytoplasm.Conclusions1.Add in in vitro cultures of 10 ng/ml and 100 ng/ml GDNF promote separation later generations before the cavity of follicles in vitro growth and development, improve the secretion E2.2.5 ng/mlGDNF can better stimulate the in vitro KGN cell proliferation, and its concentration is too large can inhibit cell proliferation.3. GDNF stimulate KGN cells in vitro, no significant change in level of estrogen progestin.4. GDNF stimulate KGN cells in vitro, can change its downstream of FSH receptor gene expression changes, increase FSH sensitivity.5. The RET GDNF receptor mRNA in KGN cells express, located in cytoplasm.4. FSH and its receptor mRNA expression has changed after 5 ng/mlGDNF stimulation, expressing quantity increased (P<0.05).5. Receptor protein of GDNF RET expression in granulosa cells, located in the cytoplasm.