Expression Of Vascular Endothelial Growth Factor And Soluble Fms-like Tyrosine Kinase Receptor 1 Under Different Oxygen Condition In The Primary Cultured Cytotrophoblasts Cells

Objective:To investigate the expression of vascular endothelial growth factor(VEGF)and soluble fms-like tyrosine kinase receptor 1(sFlt-1)under different oxygen condition at different incubation periods in the primary cultured cytotrophoblasts cells(CTs).Methods:Villi tissues of normal 6～8 weeks pregnancy were obtained under sterility condition.Cells were collected by trypsin-DNase type 1 digestion and Percoll gradient centrifugation.CTs were primarily cultured and their purities were accessed by immunocytochemistry. Primarily cultured CTs were divided into four groups:normoxic group(20%O₂),hypoxic group 1(8%O₂),hypoxic group 2(2%O₂),hypoxic and reoxygenation group(2%O₂/20%O₂),respectively.CTs in normoxic group,hypoxic group 1,hypoxic group 2 were cultured for 72h,96h,120h and 144h,respectively.CTs in hypoxic and reoxygenation group were cultured under hypoxic for 72h(2%O₂ for 72h),cultured under normoxic 24h(total 96h),48h(total 120h),72h(total 144h).Typan blue exclusion test was used to observe the vigor and calculate the viability of CTs.VEGF and sFlt-1 concentrations at different incubation periods in each culture media were measured using competitive ELISA.Results:The expression of VEGF and sFlt-1 concentrations under different oxygen tension at different incubation periods of CTs were:(1)VEGF concentrations under normoxic group,hypoxic group 1,hypoxic group 2 which were cultured for 72h,96h,120h and 144h of CTs were: normoxic group 223±32 pg/mL,271±33 pg/mL,313±41 pg/mL,361±49 pg/mL;hypoxic group 1 347±75 pg/mL,427±65 pg/mL,491±48 pg/mL,544±72 pg/mL;hypoxic group 2 382±50 pg/mL,442±49 pg/mL,503±42 pg/mL,556±70 pg/mL).VEGF concentrations of CTs under normoxic group,hypoxic group 1,hypoxic group 2 were increased with time in culture;VEGF concentrations of CTs under hypoxic group 1 and hypoxic group 2 were significantly higher than normoxic group in each culture time(P＜0.01);There wasn't a significant difference between hypoxic group 1 and hypoxic group 2(P＞0.05).(2)sFlt-1 concentrations under normoxic group,hypoxic group 1,hypoxic group 2 which were cultured for 72h,96h,120h and 144h of CTs were:normoxic group 904±231 pg/mL,1339±321 pg/mL,2013±452 pg/mL,2541±577 pg/mL; hypoxic group 1 1929±489 pg/mL,2543±785 pg/mL,3365±1004 pg/mL, 3861±1151 pg/mL;hypoxic group 2 2008±466 pg/mL,2687±675 pg/mL, 3488±948 pg/mL,4017±1105 pg/mL).sFlt-1 concentrations of CTs under normoxic group,hypoxic group 1,hypoxic group 2 were increased with time in culture;sFlt-1 concentrations of CTs under hypoxic group 1 and hypoxic group 2 were significantly higher than normoxic group in each culture time(P＜0.01);There wasn't a significant difference between hypoxic group 1 and hypoxic group 2(P＞0.05).(3)VEGF and sFlt-1 concentrations(401±55 pg/mL,2282±419 pg/mL)of CTs in hypoxic and reoxygenation group measured at 72h for hypoxic condition were obviously higher than those of normoxic group(P＜0.01),There wasn't a significant difference compared to hypoxic group 1 and hypoxic group 2(P＞0.05).(4)VEGF concentrations(362±50 pg/mL)of CTs in hypoxic and reoxygenation group measured at 24h for normoxic condition was not only obviously higher than those of normoxic group,but also significantly differrent from those of hypoxic group 1 and hypoxic group 2(P＜0.01);sFlt-1 concentration(2 086±431 pg/mL)of CTs in hypoxic and reoxygenation group measured at 24h for normoxic condition was significantly higher than that of normoxic group(P＜0.01),There wasn't a significant difference compared to those of hypoxic group 1 and hypoxic group 2(P＞0.05).(5)VEGF and sFlt-1 concentrations(350±57 pg/mL,1932±448 pg/mL;321±35 pg/mL,1831±441 pg/mL)of CTs in hypoxic and reoxygenation group measured at 48h,72h for normoxic condition were significantly lower compared to those of hypoxic group 1 and hypoxic group 2(P＜0.01)and remaining this state for 48h;but no obvious difference compared to normoxic group(P＞0.05).Conclusions:VEGF and sFlt-1 concentrations of CTs increased with time in response to hypoxic condition.There wasn't a significant difference between 2%O₂ and 8%O₂ oxygen tension in each culture time.VEGF,sFlt-1 concentrations recovered to normal level of normoxic condition after being reoxygenated for48h,72h and remaining this state for 48h.This unique response of VEGF system to oxygen tension in CTs might be closely involved in the mechanism of uterine vascular remodeling.Despite an increase in VEGF production in response to hypoxic condition,a concomitant even greater increase in sFlt-1 might override the effects of VEGF and,thereby,rather attenuate the ultimate capacity of CTs to stimulate angiogenesis.Besides,the present findings might give some help for an understanding of the pathogenesis of preeclampsia,we can take protective measures because high-level sFlt-1 will predict the genesis of preeclampsia,in order to reduce the incidence rate of preeclampsia.