The Investigation Of Angiogenesis Of Endometriosis In Vitro Cell Coculture Model

Objective1、 To establish an in vitro co-culture model of endometrial stromal cells(ESCs) and Human umbilical vein endothelial cells(HUVECs).2、 Using this model, to observe cell movement and migration of ESC and HUVEC from endometriosis(em) and non-endometriosis(con) patients, and to compare the difference of cell behaviors during cell co-cultured.3、 To investigate the impact on cell growth by β-Estradiol(E)、 Progesterone(PO) and Vascular endothelial growth factor (VEGF) during cell co-culture.Methods1、 Primary ESCs and HUVECs were isolated and cultured.2、 PDMS stamps were designed and prepared.3、 Healing test were performed to observe the difference between ESCs and HUVECs.4、 Co-culture of ESCs from endometiosis and non-endometriosis patients and HUVECs were finished. Immuno fluorescence staining was obtained to comparethe difference of cell behaviors between two groups.Results1、 With400um channels width,800um channel spacing,5x106/l density of cells, ESCs and HUVECs grew well in the channels and solid adherence was achieved within2hours.2、 In the healing test, with200um channel spacing, HUVECs healed completely withinl9hous while ESCs did not.(There was no significant difference between two kinds ESCs from endometriosis and non-endometriosis patients.)3、 Density of HUVECs in the area near ESCs was higher.4、 Growth rate of HUVECs in co-culture model was higher than that grew alone, and the effect is stronger in endometriosis group than in normal control group.(P＜0.05)5、 Impact on cell movement by β-estradiol, progesterone and VEGF in cell co-culture:1) HUVECs growth rate was higher by adding high concentration β-estradiol (2.0nmol/1), and HUVECs growth rate was slower by adding low concentration β-estradiol (0.2nmol/l)(P<0.05). Besides,cell growth rate was positively correlated with β-estradiol concentration (P<0.05)2) HUVECs growth rate was slower by adding high concentration progesterone(30nmol/1,70nmol/1), cell growth rate was negatively correlated with progesterone concentration (P<0.05)3) HUVECs growth rate was higher by adding high concentration VEGF(750ng/1), cell growth rate was positively correlated with VEGF concentration (P<0.05).Conclusion1、 Microfluidic technology has been successfully applied to established an in vitro cell co-culture modelfor angiogenesis in endometriosis. The model is stable and easy-operating.2、 HUVECs can be induced by ESCs, the effect is stronger in endometriosis group than in normal control group.3%High concentrationβ-estradiol and VEGF promote HUVECs growth, while high concentration progesterone retard HUVECs growth.4、 Low concentration β-estradiol retard HUVECs growth.