| Background and purpose:With the postponement of reproductive age,environmental pollution and living pressure increases,the incidence of infertility showed a rising trend.In recent years,the rapid development of IVF-ET and its related derivative technologies and its widespread clinical application have made many infertility patients have the opportunity to obtain offspring.The key to the success of IVF-ET is the successful implantation of the embryo.The overall pregnancy rate of assisted reproduction hovering around 40%-60%[1],but many couples continue to suffer after repeated pregnancy,the economy and the spirit are under great pressure,clinicians are also depressed.From Thomhill proposes the definition of repeated implantation failure(RIF)[2]in 2005 to Margaliothle et al.suggest that embryonic age and patient age also should be considered in later[3]the definition of RIF most scholars used in present is:After more than 3 high-quality embryos transplantation or a total number of embryos of multiple transplantation more than 10 pieces,still failing to get clinical pregnancy.RIF has become a bottleneck problem of improving the pregnancy rate in assisted reproduction.How to improve the outcome of these patients and improve the success rate of embryo implantation have become an urgent problem to be solved.The incidence of RIF in IVF-ET of Reproductive Center in Southern Hospital is about 7 to 11%[4].Quan song et al.consider the reason are several aspects as follows:Poor embryo quality,defect of transplantation,endometrial receptivity,spiritual and psychological factors and other unknown reasons.About 2/3 repeated implantation failure is due to endometrial receptivity[5,6].Therefore,it is important to study how to improve the receptivity of endometrium and its related mechanism in peri-implantation period,which has become a hotspot of RIF,and it's of great significance to improve the pregnancy rate of RIF patients.The reproductive system of women in child-bearing age has physiological angiogenesis,which is vital to the growth and development and basic function of the reproductive system[15,16],the previous study of endometrial histology found that the local physiological angiogenesis activity of endometrium is at the peak in the process of peri-implantation period is one of the most significant signs of reproductive system production in peri-implantation period.Macrophages are an ancient immune cell,which play a key role in innate and adaptive immune responses,and are the major regulatory cell in the inflammatory response[17].Macrophages have a wide range of functions,such as the phagocytosis,antigen presentation and bactericidal effect in classic immunology,but also secrete a variety of cytokines to paly different role in the different tissues and internal environment[10-12][12,18-21][12,18-21][12,18-21][12,18-21][12,18-21].In recent years,some scholars have found that macrophages,in addition to the classic immune function,there are some new features such as promoting angiogenesis and vascular remodeling and so on[22,23],which suggests that macrophages may also be involved in the formation of embryo implantation.In this study,we used rat NR8383 macrophage cell lines to study the PGE2 regulated the production of VEGF by regulating NR8383 macrophages in rats,and might further regulate its activity on angiogenesis[24].However,the mechanism of human macrophages has not been reportedIn this study,we selected human THP-1 macrophage and its role in promoting angiogenesis was taken as a starting point,though investigated the role of human THP-1 macrophages in up-regulating the expression of vascular endothelial growth factor(VEGF)protein and its regulation mechanism,to explored its related role in promoting angiogenesis,and provided experimental basis for the clinical treatments of RIF.Chapter 1 PGE2 upregulates VEGF expressions in THP-1 macrophages and its mechanismSection 1 PGE2 upregulates VEGF expressions in THP-1 macrophagesObjective:To explore the possible role of macrophages in the female reproductive system in promoting angiogenesis,we chose the THP-1 macrophages in extrocorporeal experiment,by decting the protein expression level of VEGF in THP-1 macrophages,to verify that whether PGE2 can affect THP-1 macrophages' ability to secret VEGF.Methods:1.Cell culture and inductionWe selected the well-conditioned monocyte cell lines THP-1 cells,induced to THP-1 macrophages from suspension to adherence with PMA.2.The expression of VEGF in in THP-1 macrophages treated by PGE2The well-conditioned THP-1 macrophages were treated with 0/1 ?mol/L PGE2/10p.mol/L PGE2 respectively,the protein expression of VEGF was detected by Western Blot.3.Statistical analyses were performed using SPSS version 20.0,and measurement data is expressed in mean ± standard deviation(mean ± SD).The comparison of multiple mean with analysis of variance,two-two comparisons among the means were analyzed by LSD method while equal variance,and Duimett T3 method while equal variances were wnot assumed.P<0.05 was considered statistically significant.Result:Compared with the non-treated control group,the protein express of VEGF in THP-1 treated with 1?mol/L PGE2 increased significantly(P<0.05);Compared with the non-treated control group,the protein express of VEGF in THP-1 treated with 10?mol/L PGE2 increased significantly(P<0.05);Compared with the 1?pmol/L PGE2 group,the protein express of VEGF in THP-1 treated with 10?mol/L PGE2 increased significantly(P<0.05)Conclusion:PGE2 upregulates VEGF expressions in THP-1 macrophages,and its ability was correlated with the concentration of PGE2.Section 2 The mechanism of how PGE2 upregulates VEGF expressions in THP-1 macrophagesObjective:To explore wether the receptor antagonist of PGE2(EP2 specificity-receptor antagonist--AH6809 or EP4 specificity-receptor antagonist--AH23848)or SQ22536 or H89 affect the influence on THP-1 macrophages induced by PGE2.We chose the THP-1 macrophages in extrocorporeal experiment,by decting the protein expression level of VEGF in THP-1 macrophages,to verify the mechanism of PGE2 affect THP-1 macrophages'ability to secret VEGF.Methods:1.Cell culture and inductionWe selected the well-conditioned monocyte cell lines THP-1 cells,induced to THP-1 macrophages from suspension to adherence with PM A.2.The expression of VEGF in in THP-1 macrophages treated by PGE2The well-conditioned THP-1 macrophages were treated with 10?mol/L AH6809/10?mol/L PGE2/10?mol/L AH23848/30?mol/L SQ22536/10?mol/L H89 respectively and then 10?mol/L PGE2 23 hours,the protein expression of VEGF was detected by Western Blot.3.Statistical analyses were performed using SPSS version 20.0,and measurement data is expressed in mean ± standard deviation(mean ± SD).The comparison of multiple mean with analysis of variance,two-two comparisons among the means were analyzed by LSD method while equal variance,and Duimett T3 method while equal variances were wnot assumed.P<0.05 was considered statistically significant.Result:AH6809 can inhibit PGE2 enhancing the protein expression of VEGF in THP-1 macrophages,while AH23848 can'n.Fuether more,SQ22536 and H89 can inhibit PGE2 enhancing the protein expression of VEGF in THP-1 macrophages.Conclusion:PGE2 upregulates VEGF expressions in THP-1 macrophages,and its ability was correlated with the concentration of PGE2 via EP2 and cAMP-PKA signaling pathway.Chapter 2 PGE2 affects the THP-1 macrophages' ability in promoting angiogenesisSection 1 PGE2 affects the THP-1 macrophages' ability in promoting HUVECs migrationObjective:As shown previously,PGE2 can increase the protein expression of VEGF in THP-1 macrophages via EP2 and cAMP-PKA signaling pathway,which indicating that PGE2 may promote angiogenesis.Thus,we used extracorporeal cytology experiment—transwell,processing HUVECs with different treatment processed THP-1 macrophages cultivation supernate,observeing the number of HUVECs migration,verify wether PGE2 can enhance the THP-1 macrophages promoting HUVECs migration and its further machanism.Methods:1.Cell culture and inductionWe selected the well-conditioned monocyte cell lines THP-1 cells,induced to THP-1 macrophages from suspension to adherence with PMA.2.The migration ability of HUVECs induced by PGE2d was detected by Transwell The well-conditioned THP-1 macrophages were treated as the following groups:Non-treatment group:THP-1 macrophages with no treatment 1?mol/L PGE2 group:THP-1 macrophages were treated with 1?mol/L PGE2 for 24 hous.10?mol/L PGE2 group:THP-1 macrophages were treated with 10?mol/L PGE2 for 24 housAH6809+ PGE2 group:THP-1 macrophages were treated with 10?mol/L AH6809 for 1 hour before 10?mol/L PGE2 for 23 housAH23848+ PGE2 group:THP-1 macrophages were treated with 10?mol/L AH23848 for 1 hour before 10?mol/L PGE2 for 23 housSQ22536+ PGE2 group:THP-1 macrophages were treated with 30?mol/L AQ22536 for 1 hour before 10?mol/L PGE2 for 23 housH89+ PGE2 group:THP-1 macrophages were treated with 10?mol/L H89 for 1 hour before 10?mol/L PGE2 for 23 housAfter washed the macrophages with PBS and further cultured for 8 hours with serum-free medium,the migration number of HUVECs was dectected by Transwell to analyze the ability of PGE2 affects the THP-1 macrophages' in promoting HUVECs migration.3.Statistical analyses were performed using SPSS version 20.0,and measurement data is expressed in mean ± standard deviation(mean ± SD).The comparison of multiple mean with analysis of variance,two-two comparisons among the means were analyzed by LSD method while equal variance,and Duimett T3 method while equal variances were wnot assumed.P<0.05 was considered statistically significant.Result:The number of migrating HUVECs in 1?mol/L PGE2 group/10?mol/L PGE2 group were higer than the number of the non-treated control group(P<0.05);The number of migrating HUVECs in 10?mol/L PGE2 group were higer than the number of 1?mol/L PGE2 group(P<0.05);the number of migrating HUVECs in AH6809+PGE2 group/SQ22536+ PGE2 group/H89+ PGE2 group were lower than the number of 10?mol/L PGE2 group(P<0.05),but there is no statistical difference between the number of migrating HUVECs in AH23848+ PGE2 group and 10?mol/L PGE2 group.Conclusion:1.PGE2 can enhance the ability of THP-1 macrophages in promoting HUVECs migration.After PGE2 processing,the THP-1 macrophages ability in promoting HUVECs migration was enhanced which involve the concentration of PGE2.It is consistent with the result of Western blot.2.The mechanism of PGE2 affect the ability of THP-1 macrophages in promoting HUVECs migration The PGE2 specificity receptor-EP2 antagonist—AH6809/adenylyl cyclase inhibitor--SQ22536/PKA inhibitor H89 can suppress PGE2 to enhance the migration of HUVECs,which indicade that PGE2 affect the migrating ability of HUVECs induced by THP-1 macrophages via EP2 and cAMP-PKA signaling pathway.Section 2 PGE2 affects the THP-1 macrophages' ability in promoting HUVECs forming vesselsObjective:As shown previously,PGE2 can increase the protein expression of VEGF in THP-1 macrophages via EP2 and cAMP-PKA signaling pathway,which indicating that PGE2 may promote angiogenesis.Thus,we used extracorporeal cytology experiment—Matrigel,processing HUVECs with different treatment processed THP-1 macrophages cultivation supernate,observeing the size of HUVECs forming vessels,to verify wether PGE2 can enhance the THP-1 macrophages promoting HUVECs forming vessels and its further machanism.Methods:1.Cell culture and inductionWe selected the well-conditioned monocyte cell lines THP-1 cells,induced to THP-1 macrophages from suspension to adherence with PMA.2.The migration ability of HUVECs induced by PGE2d was detected by Transwell The well-conditioned THP-1 macrophages were treated as the following groups:Non-treatment group:THP-1 macrophages with no treatment 1?mol/L PGE2 group:THP-1 macrophages were treated with 1?mol/L PGE2 for 24 hous10?mol/L PGE2 group:THP-1 macrophages were treated with 10?mol/L PGE2 for 24 housAH6809+ PGE2 group:THP-1 macrophages were treated with 10?mol/L AH6809 for 1 hour before 10?mol/L PGE2 for 23 housAH23848+ PGE2 group:THP-1 macrophages were treated with 10?mol/L AH23848 for 1 hour before 10?mol/L PGE2 for 23 housSQ22536+ PGE2 group:THP-1 macrophages were treated with 30?mol/L AQ22536 for 1 hour before 10?mol/L PGE2 for 23 housH89+ PGE2 group:THP-1 macrophages were treated with 10?mol/L H89 for 1 hour before 10?mol/L PGE2 for 23 housAfter washed the macrophages with PBS and further cultured for 8 hours with serum-free medium,the size of HUVECs forming vessels was dectected by Matrigel to analyze the ability of PGE2 affects the THP-1 macrophages' in promoting HUVECs forming vessels.3.Statistical analyses were performed using SPSS version 20.0,and measurement data is expressed in mean ± standard deviation(mean ± SD).The comparison of multiple mean with analysis of variance,two-two comparisons among the means were analyzed by LSD method while equal variance,and Duimett T3 method while equal variances were wnot assumed.P<0.05 was considered statistically significant.Result:The size of HUVECs forming vessels in 1?mol/L PGE2 group/10?mol/L PGE2 group were higer than the number of the non-treated control group(P<0.05);the size of HUVECs forming vessels in 10?mol/L PGE2 group were higer than the number of 1?mol/L PGE2 group(P<0.05);the size of HUVECs forming vessels in AH6809+PGE2 group/SQ22536+ PGE2 group/H89+ PGE2 group were lower than the number of 10?mol/L PGE2 group(P<0.05),but there is no statistical difference between the size of migrating HUVECs in AH23848+ PGE2 group and 10?mol/L PGE2 group.Conclusion:1.PGE2 can enhance the ability of THP-1 macrophages in promoting HUVECs forming vessels.After PGE2 processing,the THP-1 macrophages ability in promoting HUVECs forming vessels was.enhanced which involve the concentration of PGE2.It is consistent with the result of Western blot.2.The mechanism of PGE2 affect the ability of THP-1 macrophages in promoting HUVECs forming vessels.The PGE2 specificity receptor-EP2 antagonist—AH6809/adenylyl cyclase inhibitor--SQ22536/PKA inhibitor H89 can suppress PGE2 to enhance the ability of forming vessels of HUVECs.It is consistent with the result of Western blot. |
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