Effect Of Hydroxy Safflor Yellow A On The Proliferation And The Expression Of Cytokine Of The Cultured Eutopic Endometrial Cells From Endometriosis Patients

Objective: Different concentrations of HSYA interfered in vitro endometriosis (Endometriosis, EMs) eutopic endometrial cells, the growth and proliferation of eutopic endometrial cells were measured by Blue tetrazolium method (MTT) test.The expression of the VEGF protein and the bFGF protein in the medium induced by HSYA were analyzed by enzymelinked immunosorbent assay (ELISA).To explore the feasibility of HSYA for the treatment of endometriosis.Methods:1 The study subject:1.1 The study group: patients who undergoing laparotomy or laparoscopy for endometriosis in our hospital, selected from October 2009 to February 2010(proliferative phase were pathologically confirmed), a total of 15 cases (mean age [±SD] 38.13±7.26years old).1.2 The control group: Selected the patients in the same period in our hospital with septate uterus, ovarian dysembryoma or simple cyst and without pelvic endometriosis (normal proliferative phase endometrium were pathologically confirmed), a total of 14 cases (mean age [±SD] 34.92+8.11years old).All of the women in the two groups had normal menstrual cycles, nearly 6 months without taking steroids and immunosuppressive drugs history and the history of IUD placement. The patients with endometrial polyps, acute or chronic pelvic inflammatory diseases, breast cancer, endometrial cancer and other gynecological malignancies were excluded. All of them had not endocrine, immune and metabolic diseases and high blood pressure, heart disease, kidney disease etc. Samples were taken by the patient's consent, and signed informed consent.2 Specimen collecting:The endometrium samples were scraped and placed in ice- bath 1:1 DMEM/F12 medium containing penicillin and streptomycin, sent to the laboratory to culture in 2 hours.3 Experiment methods:3.1 Cell cultureEndometrial tissue was rinsed, separated, digested, filtrated.The cells were cultured in flasks, which contained DMEM/F-12 medium supplemented with 10% NBCS, penicillin and streptomycin at 37°C in humidified atmosphere of 5% CO2 in air. After 24 hours replaced the medium, discarded the no adherent cells and red blood cells, then changed the medium every other day. Under the Inverted microscope observed cell morphology and growth conditions.When the cells covered 80% of the flasks wall, were digested by trypsin, and plated in flasks in the same agent to culture, changed the medium every other day. The ECs were identified byimmunohistochemical staining, with markers specific to ESCs (Vimentin), epithelial cells (Cytokeratin).3.2 Cell proliferation assay:3.2.1 The normal growth curve:Took the first 2-3 generations ECs to seed in 96-microwell plates, and set a withered well. And incubated for 24h,48h,72h, 96h,120h,144h, added MTT and Dimethyl Sulfoxide per well. Enzyme-linked immunosorbent detector determined the cell absorbance (OD value) at 492nm wavelength. Draw cell growth curve by Microsoft Excel software.3.2.2 The cell growth curve after the intervention of the medicinesTook the first 2-3 generations ECs to seed in 24-microwell plates, after 24h incubation, the cells were washed by PBS. Then added HSYA and GnRHa, cultured for 24h, 48h, 72h. Each group set up five wells, and a control group. Cultured for an additional 24h, 48h, 72h. The absorbance values were determined at 492 nm wavelength. Microsoft Excel software draw cell growth curve.3.3 Measurement of VEGF and bFGF by ELISATook the first 2-3 generations ECs to seed in 24-microwell plates, and set a control well and incubated with 10% NBCS FD medium for 24 h, subsequently washed and incubated with medium supplemented with 2% NBCS for an additional 24 h under basal conditions, after the addition of serum-free FD with different concentrations of GnRHa and HSYA for 48 h. Each group set up three wells. The control group did not add any drugs. Culture supernatants were collected and kept at -20°C to be measured. The VEGF and bFGF concentration in culture supernatants was quantitated using a commercial ELISA kit specific for human VEGF/bFGF.Results:1 Cell growth condition:Endometrial cells from EMs patients were successfully cultured, and the achievement ratio was 93.3% (14/15). The achievement ratio of the normal endometrial cells was 92.9% (13/14). The viability was at least 90%.2 Identification of cells in immunocytochemistry①Endometrial stromal cells stainned positive for vimentin, cell cytoplasm showed brown.②Endometrial glandular epithelial cells stainned positive for Cytokeratin, cell cytoplasm showed brown.③Negative control:PBS as antibody,Vimentin and Cytokeratin stainned negative。Inverted microscope observed the cells. Endometrial cells began to adhere after 0.5h and 24h later basically completed adherent and began to grow. About 2-4days cells grew to speed up, entered the platform period, and the cells linked tightly with each other.3 The effect of HSYA and GnRHa on the proliferation of eutopic endometrial cells3.1 The effect of HSYA on the proliferation of in vitro cellsAfter 24h, HSYA could inhibit the proliferation of eutopic endometrial cells from women with endometriosis, but no statistical significance.After 48h, 72h, HSYA significantly inhibited cell growth in a dose dependent manner, the concentration of HSYA were higher the smaller of the OD value (proportional to cell number).HSYA significantly inhibited the proliferation of the ECs with the concentration and time increased.3.2 The effect of GnRHa on the proliferation of in vitro cells After 24h, GnRHa could inhibit the proliferation of eutopic endometrial cells from women with endometriosis, but no statistical significance.After 48h, 72h, GnRHa significantly inhibited cell growth in a dose dependent manner. GnRHa significantly inhibited the proliferation of the ECs with concentration and time increased.3.3 The effect of HSYA and GnRHa on the proliferation of in vitro cellsAfter 24h, both drugs inhibited the proliferation of the ECs but no significant difference.After 48h, the HSYA20,2,0.2μg/ml and GnRHa10,1,0.1μg/ml group compared respectively, GnRHa had stronger inhibition on cells in vitro than HSYA. HSYA 20μg/ml group inhibited the proliferation of the ECs stronger than the GnRHa 1μg/ml group. HSYA 2μg/ml group inhibited the proliferation of the ECs stronger than GnRHa 0.1μg/ml group.After 72h, the HSYA 20,2,0.2μg/ml and GnRHa10,1,0.1μg/ml group compared respectively, GnRHa had stronger inhibition on cells in vitro than HSYA. HSYA 20μg/ml group inhibited the proliferation of the ECs stronger than GnRHa 0.1μg/ml group. HSYA 2μg/ml group inhibited the proliferation of the ECs stronger than the GnRHa 0.01μg/ml group.4 In spite of both drugs inhibited normal endometrial cells, had no significant distinction.5. The expression of cytokine in cell culture supernatant5.1 The expression of VEGF in supernatant was significantly decreased after the treatment of HSYA and GnRHa.Compared With the control group without drugs, various concentrations groups all significantly inhibited the expression of VEGF in a dose dependence manner. Comparison of the inhibition of the two drugs, HSYA 20, 0.2μg/ml groups had stronger inhibition than GnRHa 0.1,0.01μg/ml groups.Thus, although the inhibition of GnRHa on the expression of VEGF stronger than HSYA, but in a certain concentration range HSYA was stronger than the GnRHa.5.2 The expression of bFGF in supernatant was significantly decreased after the treatment of HSYA and GnRHa.Compared With the control group without drugs, HSYA 20,2μg/ml groups and GnRHa10, 1μg/ml groups significantly inhibited the expression of bFGF in a dose dependence manner.Comparison of the inhibition of the two drugs, HSYA 20,2μg/ml groups inhibited the expression of bFGF significantly stronger than the GnRHa 10,1μg/ml groups. Thus HSYA had stronger inhibition on the expression of bFGF than GnRHa.Conclusion:1 An in vitro model of endometrial cells successfully established.2 Endometrial cells included stromal cells and glandular epithelial cells.Stromal cells stained positive for vimentin, epithelial cells stained positive for cytokeratin. In the cell suspension, the stromal cells were single, round cells. The gland epithelial cells were multiple cell clusters, thyrsiform.3 Endometrial cells began adherent after 0.5h and about 24h later basically completed adherent and began to grow. About 2-4days cells grew speed up, after 5-6 days entered the platform period, and the cells linked tightly with each other.4 HSYA and GnRHa both significantly inhibited the proliferation of the ECs in a dose-time dependence manner. Comparison of HSYA at the same level, GnRHa had stronger inhibition, but in a certain concentration range HSYA inhibited the proliferation of the ECs than GnRHa stronger.5 Both HSYA and GnRHa inhibited the expression of VEGF and bFGF in cell culture supernatant dose-dependent. GnRHa inhibited the expresstion of VEGF stronger than HSYA. HSYA inhibited the expresstion of bFGF stronger than GnRHa.6 Both drugs could inhibited normal endometrial cells, had no statistically significant.HSYA inhibited the proliferation of endometrial cells from EMs patients may be through its down regulation the expression of VEGF and bFGF implementation. Prompted HSYA may be a potential drug for the treatment of endometriosis, to provide a new theoretical basis for the theropy of endometriosis.