Study On The Mechanism Of Inflammatory Effect Of Maternal - Fetal Interface In Pregnancy And Its Related Diseases

Objectives:(1) To compare the expression of Interleukin 27(IL-27) and its specific receptor WSX-1 between normal pregnant women and preeclampsia(PE) patient;(2) To identify the inflammatory effect triggered by IL-27 in human trophoblast cells(HTR-8/SVneo);(3) To investigate the possible role of IL-27 and its downstream signaling pathways in the pathogenesis of preeclampsia and identified the underlined mechanisms which provide new potential therapeutic targets for preeclamosia.Methods:(1) Differential expression of IL-27 in serum between normal pregnancy women and PE patients was detected by ELISA;(2) Differential expression of IL-27 and its specific receptor WSX-1 in placental tissues between normal pregnancy women and PE patients was detected by immunohistochemistry;(3) qRT-PCR and Western Blotting were used to identify the expression of WSX-1 and gp130 which were receptors of IL-27 in trophoblast cells of HTR8/SVneo;(4) RT-PCR screened of the downstream inflammatory factors after IL-27 treatment in HTR8/SVneo cells;(5) Applied RT-PCR and Western Blotting respectively verify the target downstream inflammatory mediators release after treatment with IL-27 both at RNA level and protein level;(6) ELISA was used to screen the classical inflammatory factors which could synergy with IL-27 to trigger excessive inflmmation;(7) To screen the downstream signaling pathways after stimulated with IL-27 in HTR8/SVneo cells, and implement Western Blotting to verify at protein level.Results:(1) There was no difference in the expression of IL-27 in the serum between normal pregnancy women and PE patients;(2) Immunohistochemistry confirmed that IL-27 and its specific receptor WSX-1 can be expressed in placental tissue, and the expression in PE group is higher than that in normal pregnancy group;(3) WSX-1 and gp130 were both expressed in HTR8/SVneo cells;(4) IL-27 mainly induced excessive inflammation by stimulating the release of inflammatory mediators CXCL10 and IL-6 in a time dependent manner;(5) IL-27 could significantly enhance the release of IL-6 and CXCL10 synergy with TNF-α;(6) IL-27 through the activation of phosphatidylinositol 3-OH kinase-AKT(PI3K), p38 mitogen activated protein kinases(MAPK) and Janus kinase and signal transducer and activator of transcription(JAK / STAT) signaling pathway increased downstream inflammatory factors of CXCL10 and IL-6.Conclusion:(1) IL-27 stimulated the release of CXCL10 and IL-6 to trigger excessive inflammation in HTR8/SVneo cells which participate in the occurrence of PE.(2) TNF-α could synergy with IL-27 to cause the excessive inflammation;(3) Through the activation of PI3K-AKT, p38 MAPK and JAK/STAT signaling pathways, IL-27 causes the release of CXCL10 and IL-6, resulting in excessive inflammation;(4) The excessive inflammation was suppressed after respectively applied LY294002, SB253580 and AG490 which were the specific inhibitors for PI3K-Akt, p38 MAPK and JAK/STAT. This may provid potential new ideas and targets for the treatment of preeclampsia.Objectives:(1) To compare the chemotactic effect of whole fetal membrane(amnion, chorion and decidua) homogenate and supernatant on peripheral leukocytes between TNL(term none labour) pregnant women and TL(term in labour) pregnant women.(2)To screen activated chemokine candidates at whole fetal membrane(FM) homogenate and supernatant.(3) To identify the specific targeting chemokine candidates depend on their chemotactic ability which may provide new potential diagnostics and therapeutic targets for preterm birth.Methods:(1) To establish an in vitro culture model of FM tissues;(2) LMA(Leukocyte migration assay) was applied to compare the chemotactic ability of FM homogenate and supernatant between TNL and TL,and use the specific antibody to mark migrated leukocytes then detected by flow cytometry to distinguish the subpopulation of leukocytes, and compared between TNL and TL;(3) FM homogenate and supernatant both from TNL and TL groups were separately applied Proteome Profiler to screen the activated chemokines;(4) q RT-PCR and Bio-Plex TM validated the output of these activated chemokine candidates at RNA and protein levels;(5) Bio-PlexTM quantified the concertration of those chemokine candidates in serum;(6) LMA was used to verify the chemotaxis of human target chemokines recombinant protein at physiological dose, and to test whether their chemotaxis decreased after using the specific neutralizing antibody.Results:(1) FM culture model in vitro has been successful established;(2) The attracted total peripheral leukocytes were increased in TL compared to TNL both in FM homogenate and supernatant, especially for granulocytes and monocytes(P<0.05), whereas the percentage of subspopulation was not alter(P>0.05).(3) Proteome Profiler identified 6 chemokine candidates included CXCL1, CXCL8, CXCL10, CCL2, CCL5 and CCL21;(4) CXCL1, CXCL8 and CCL21 significantly elevated in TL compared to TNL both in FM homogenate and supernatant at RNA and protein levels(P<0.05).(5) Unfortunately, the concentration of these chemokine candidates in serum were found no alter between TNL and TL(P>0.05);(6) Human recombinant protein of CXCL1 and CXCL8 attracted peripheral leukocyte in a dose-dependent manner at physiological dose of FM supernatant(p>0.05), and the chemotactic activity of FM supernatant significantly suppressed after stimulated with CXCL1 and CXCL8 specific neutralization antibodies(p>0.05), however, this effect was not observed in FM homogenate(p<0.05).Conclusion:(1) We successfully established an in vitro model to mimic fetal membrane mediated peripheral leukocyte migration during parturation contribute to further understand the physiological mechanism of parturation and preterm birth;(2) The number of infiltration leukocytes increased at the fetal membrane during labour, especially for granulocyte and monocytes, may play an important role in the process of birth initiation.(3) CXCL1 and CXCL8, which are confirmed by comprehensive and systematic screening, play an important role in mediating leukocyte infiltration of fetal membranes, which may be a new target for the diagnosis and treatment of preterm labor.(4) There are still some unknown chemokines in fetal membranes to initial of labor, except CXCL1 and CXCL8.