The Expression Of GnRHâ…¡ Protein In The Endometriotic Patients And The Effect On Endometrial Etromal Cells In Vitro

Endometriosis (short for EMs) is one common and frequently-occurring disease in women of reproductive age. Its pathogenesis is unclear,recurrent rate is high,treatment is thorny, these make EMs become difficult and hot research. Therefore, to study the pathogenesis of EMs and to find effective treatment methods,which becomes an urgent problem. In recent years, the combination of gonadotropin-releasing hormone analogues (GnRHa) and laparoscopic surgery in the treatment of EMs are sure to be useful and effective. Recent study found that:Ⅱ-type gonadotropin-releasing hormone (GnRHⅡ) can inhibit cell growth in some tumors of the reproductive system,such as ovarian cancer,breast cancer and prostate cancer, and had stronger anti-proliferative effects than GnRH I in endometrial cancer and ovarian cancer cells. Endometriosis is a hormone-dependent disease, which has some biological behaviors of malignant tumor. Therefore, our article attempts to study the expression of the GnRH II in patients with endometriosis and its effects on the eutopic and ectopic endometrial stromal cells in vitro culture in the following three parts:(1). Using the method of SP immunohistochemistry to investigate whether there is expression of GnRHⅡprotein in ectopic and eutopic endomentrium from patients with endometriosis, and normal endometrium was used as control, to study whether there are differences in their expression, as well as whether the expression has cyclical changes in the these endometriums. (2) The endometrial stromal cells were isolated,cultured and identified from ectopic and eutopic endometrium tissues In vitro, then different concentrations of GnRH II (0,10-10M,10-8 M,10-6M) were added in the stromal cells in vitro culture, to detect the occurrence of apoptosis in these endometrial stromal cells with the method of Hoechst staining and flow cytometry test, in order to investigate whether GnRHⅡhas a direct inducing apoptosis effect on cultured endometrial stromal cells in vitro; (3) Different concentrations of GnRHⅡ(0,10-10M,10-8M,10-6M) were added in the stromal cells in vitro culture, and compared to GnRHⅠ, to detect the changes of concentrations for VEGF; and to compare these cells proliferation inhibitory effects by analysing cells inhibitory rate in endometrial stromal cell in vitro, so as to investigate whether GnRHⅡhas antiproliferative effects on the endometrial stromal cell in vitro. In short, to investigate the expression of endogenous GnRHⅡprotein whether has relations with the pathogenesis of endometriosis, and exogenous GnRHⅡwhether can be applied to clinical treatment of endometriosis and its possible treatment mechanisms in the patients with EMs, which providing new experimental and theoretical basis. About the above-mentioned serial studies, It has not been reported at home and abroad. Objective:To study the expression of GnRHⅡprotein in patients with-and without-endometriosis and the relation to the menstrual cycle.Methods:Thirty ectopic and thirty eutopic biopsy endometrial specimens were obtained from women with untreated endomentriosis and 40 controls were studied, which came from March 2007 to June 2008 in the Second Xiangya Hospital of Central South University. The expression of GnRHⅡprotein were tested from the ectopic,eutopic,and normal endometrium by immuneohistochemistry SP methods, and compared them.Results:1. The expression of GnRHⅡprotein positive immune staining is located in cytoplasm of endometrial stromal cells and gland cells in the all specimens.2 The expression of GnRHⅡprotein was lower in ectopic endometrium than eutopic endometrium of women with endometriosis (P<0.05),and were lower in the eutopic and ectopic endometrium with endometriosis than the nomal endometrium of women without endometriosis (P<0.05).3. The expression of GnRHⅡprotein was obvious higher in secretory phase than that of in hyperplasia phase in normal endometrial (P<0.05),especially in early and middle secretory phase(P<0.05), but there was no significant difference in eutopic and ectopic endometrium from endometriosis in the menstrual cycle(P>0.05). Conclusion:1. The positive expression of GnRHⅡin the ectopic,eutopic,and normal endometrium, and its expression was related to the menstrual cycle in normal not in endometriotic endometrium, suggests:it may play physiological and pathological roles in above endometrium.2. The lower expression of GnRHⅡin the ectopic,eutopic endometrium (P<0.05) may cause endometriosis.3. The lower expression of GnRHⅡin the eutopic endometrium suggests endometriosis may be retated to eutopic endometrial abnormal character. Objective:To culture the eutopic and ectopic endometrial stromal cells from patients with endometriosis and identify them in vitro; Then, To study the effect of GnRH II on these cells apoptosis in cultured endometrial stromal cells in vitro.Methods:First, thirty ectopic and thirty eutopic biopsy endometrial specimens were obtained from women with untreated endomentriosis, the stromal cells were separated,cultured from the grandular epithelium, then identified in vitro; Second, to detect the cell apoptosis ratio after different concentration (0,10-10 M,10-8 M,10-6 M) GnRH II were used in these stromal cells In vitro by Hoechst staining and flow cytometry test.Results:1. Eutopic endometrial stromal cells of survival rate was 83.33%,ectopic endometrial stromal cells of survival rate is 66.67% by the method of trypsin,collagenase,mesh filtration and centeifugation.2. The ectopic endometrial stromal cells of survival rate(%):the red site is 66.67%, the black site is 10.00%.3. In vitro culture, the apoptosis cell of endometrial stromal cells can be observed after GnRH II treatment (0,10-10 M,10-8 M,10-6 M), The Hoechst dye showed that eutopic and ectopic endometrial stromal cells can be induced apoptosis:cell shrinkage,nucleus condensation,and sometimes apoptopic body can be seen. The rate of apoptosis(%) is (5.78±0.53,5.79±0.66), (18.62±2.59,31.30±2.93), (47.41±3.57, 59.28±4.25), (66.16±5.46,79.43±6.42), There were statistically significant differences among different concentration experimental groups (P<0.05), the apoptosis rate of ectopic endometrial stromal cells was higher than eutopic endometrial stromal cells (P<0.05),and were dose-dependent.4. In vitro culture, the apoptosis cell of endometrial stromal cells can be observed after GnRHⅡtreatment (0,10-10 M,10-8 M,10-6 M), the flow cytometry test results showed that eutopic and ectopic endometrial stromal cells can be induced apoptosis. The rate of apoptosis (%) is (5.87±0.59,5.99±0.78), (19.30±3.28,33.40±3.97), (46.30±5.32, 62.41±6.74), (69.70±8.77,83.30±9.42), There were statistically significant differences among different concentration experimental groups (P<0.05), the apoptosis rate of ectopic endometrial stromal cells was higher than eutopic endometrial stromal cells (P<0.05),and were dose-dependent.Conclusion:1.Highly purified eutopic and ectopic endometrial stromal cells were seperated and cultured successfully by the method of trypsin,collagenase,mesh filtration and centeifugation, which provides a successful cell model and expremental basis for the pathogenesis and drug therapy of EMs.2. The one of successful culture of ovarian stromal cells for EMs was related to its site.3. Exogenous GnRH II can increase stromal cells apoptosis in dose-dependent in vitro for the patients with EMs, and the ectopic was higher than the eutopic, which suggests GnRH II may play an important role in treatment EMs by inducing cell apoptosis. Objective:1. To study the effects of GnRHⅡon the secretion of VEGF concentration by eutopic and ectopic endometrial stromal cells cultured in vitro, and contrast with GnRH I, which may provide theoretical basis for exploring GnRH II new treatment for EMs.2. Different concentrations of GnRH II were added in the cultured stromal cells in vitro, and compared to GnRH I, to detect the cell proliferation inhibitory rates of GnRH II and GnRH I on the endometrial stromal cells in vitro, and compare and analyse them.Methods:1. The above eutopic and ectopic stromal cells in vitro were divided into three groups:(1) treated by 10-10M,10-8M,10-6M GnRHⅡ; (2) treated by 10-10 M,10-8 M,10-6M GnRH I; (3) control group, not treated by GnRH. ELISA test was used to measure the concentration of VEGF protein in the medium of above three groups.2. The above eutopic and ectopic stromal cells in vitro were divided into three groups:(1) treated by 10-10M,10-8M,10-6M GnRHⅡ; (2) treated by 10-10M,10-8M,10-6M GnRH I; (3) control group, not treated by GnRH,and then were continued to culture for 24 hours,48 hours,72hours. MTT method was used to detect survival cell, and calculated to compare the inhibitory rate of stromal cells in vitro.Results: 1. There is no difference between the VEGF protein concentration by eutopic and ectopic stromal cells secreting in the medium after being cultured for 48 hours in vitro (P>0.05)2.10-10M,10-8M,10-6M GnRHⅡcan dose-dependent reduce VEGF protein concentration by endometrial stromal cells secreting (P<0.01) and the inhibitory to endometrial stromal cells is stronger than GnRH I (P<0.05).3.The inhibitory effect of GnRH II on VEGF in ectopic stromal cells is stronger than that of in eutopic stromal cells (P<0.01)4.The eutopic and ectopic endometrial stromal cell was cultured and treated with gradual high concentrations of GnRH II (10-10M,10-8M,10-6M) for 24 hours,for 48 hours,for 72 hours respectively. The inhibitory rate(%) of cell proliferation in the same endometrial stromal cell with different gradual high concentrations of GnRHⅡat the same time was gradually increased, significant difference was observed(P<0.05); the cell inhibitory rate(%) of the same endometrial stromal cell with same concentrations of GnRH II at different time was gradually increased, significant difference was observed(P<0.05); the cell inhibitory rate of ectopic endometrial stromal cell was higher than that of eutopic endometrial stromal cell, significant difference was observed (P<0.05).5.The eutopic and ectopic endometrial stromal cell was cultured and treated with gradual high concentrations of GnRH I (10-10M,10-8M,10-6M) for 24 hours,for 48 hours,for 72 hours respectively. The inhibitory rate(%) of cell proliferation in the same endometrial stromal cell with different gradual high concentrations of GnRH I at the same time was gradually increased, significant difference was observed.(P<0.05),the cell inhibitory rate of the same endometrial stromal cell with same concentrations of GnRH I at different time was gradually increased, significant difference was observed(P<0.05),in dose and time dependent manner, significant difference was observed(P<0.05), the cell inhibitory rate(%) of ectopic endometrial stromal cell was higher than that of eutopic endometrial stromal cell, significant difference was observed (P<0.05).6. GnRHⅡhad higher inhibitory rate(%) of cell proliferation in endometrial stromal cell in vitro than GnRH I, significant difference was observed (P<0.05).Conclusions:1.Ectopic stromal cells cultured in vitro can secrete VEGF, which has no difference with the eutopic stromal cells.This may play an important role in the formation and development of EMs.2.GnRHⅡcan reduce VEGF protein secreted by endometrial stromal cell cultured in vitro in dose-dependent manner, and the inhibition is stronger than GnRHⅠ.which may provide theoretic basis for exploring new treatment for EMs.3.GnRHⅡhad more antiproliferative effects on endometrial stromal cell than GnRH I analogues (goserelin) in vitro,especially in ectopic endometrial stromal cells, in dose-and time-dependent manner, which may provide a new theory for researching new drug for EMs.