| Background and Objective:Uterine leiomyoma is a common gynecological diseases,but its pathogenesis is not clear. A large number of studies have shown close relationship between estrogen and uterine leiomyoma. Traditional nuclear estrogen receptor (ER) alpha beta ER, as a transcription factor has been widely accepted, but a lot of research showed that there were no significant difference of estrogen in cycle blood between leiomyoma and normal woman. This suggests that occurrence of uterine leiomyoma may associated with the occurrence of local estrogenic difference. Transmembrane G protein coupled receptors (GPR30)(G protein-coupled receptors, G protein coupled receptor) molecular weight of about40KD, the gene was mapped to chromosome7p22, mainly located in the cytoplasm and the endoplasmic reticulum, is another type of transmembrane signal receptor protein which can activate extracellular signal-regulated kinase (ERK-1/2) GPR30has the four subunit:α,β,γ,8.A number of studies have confirmed GPR30with estrogen specific combination of rapid estrogen mediated effects on expression,this may be related to local high estrogen effect. This research hopes to further exploration the relationship between GPR30and uterine leiomyoma. Methods:(1) Draw materials, uterine leiomyoma primary cell culture, the successful culture of7-8days aftersubculture.(2) Leiomyoma cell identification, application of HE staining of leiomyoma cells, identification of cell size, morphology observation, growth characteristics and arrangement and the inverted phase contrast microscope.(3) Analysis of the expression of GPR30in uterine leiomyoma cells in the immunefluorescence method.(4) Western blotting (Western Blot) detection of GPR30expression in uterineleiomyoma cells.(5) Western blotting (Western Blot) detection of differences in uterine leiomyoma cells in untreated group of uterine leiomyoma cells and the addition of estradiol in cultured after24h and48h,the expression of Erkl/2in cells.(6) Western blotting (Western Blot) detection of normal uterine leiomyoma cells and GPR30-siRNA transfected of uterine leiomyoma cells in cultured after24h and48h,the expression differences of Erkl/2in cells.(7) Using the3-4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromid(MTT) method and by numerical time and optical density570(OD570) drawing cell growthcurve.Results:(1) After1days of culture, most of the cells have attached to the wall. Liquid change after3days of adherent cells fully extended. The majority of spindle, cells extended longer neurites and in contact with each other, some areas were the typical "peak valley" structure, cultured7-8days cells covered the bottom of the bottle. And identified as smooth muscle cells.(2) The expression of GPR30protein of Western Blot detection in human uterine leiomyoma and normal uterine smooth muscle tissues, the results showed that GPR30in the uterine leiomyoma and normal myometrium are expressed.(3) The Western Blot show in the role of estrogen, the expression of Erkl/2in uterine leiomyoma cells than in untreated group of normal human uterine leiomyoma cells was significantly increased by GPR30. After GPR30by siRNA interference, the expression of Erkl/2in smooth muscle cells of uterine leiomyoma cells interference significantlylower than that of the untreated group.(4) The cell growth curve analysis showed, in estrogen stimulation, growth and empty vector control cells increased significantly (OD values increase), GPR30interference group cell growth activity and survival rate decreased significantly.Conclusions:GPR30is highly expressed in cytoplasm of leiomyomas cells and may introduce E2participate in the proliferation of cellular leiomyoma of uterus by ERK signal pathway. |
