| Preeclampsia is a pregnancy disorder with sudden onset of maternal hypertension and proteinuria, and accompanied by systemic multiple organ damage. Severe cases may go to death due to convulsions, cerebral hemorrhage, pulmonary edema, heart failure, disseminated intravascular bleeding. Although there are a lot of studies on preeclampsia, the pathogenesis of preelcampsia is still unclear. Due to the lack of specific biological indicators and effective interventions, the preeclampsia is still a major contributor to the maternal and neonatal mortality. Chemokine CXCL12, also known as stromal cell derived factor 1, expressed in many tissues, such as heart, liver, kidney, brain and bone marrow and so on. CXCL12 is the key factor of the hematopoietic stem cell mobilization and homing, as well as play an important role in immune cell activation, migration, invasion and metastasis of tumor cells, and embryonic growth and development. Previous study had been confirmed that CXCR4 is the only receptor of CXCL12, However there is more and more evidence show that CXCR7 is also the recepter of CXCL12. CXCR7 could interact with CXCR4 and then formed heterodium, which involved in the recruitment of ?-arrestin, and activation of ?-arrestin-linked signaling pathways. Moreover, CXCR7 could bound to CXCL12 independently and then mediated the transmission of signaling pathways. CXCR7 functions as a specific scavenger for CXCL12. CXCL12 binds the chemokine receptor CXCR7 with an higher affinity than CXCR4.Our previous work focused on the expression and function of CXCL12/ CXCR4/CXCR7 axis at the maternal-fetal interface of human first-trimeter. Based our the previous work, an economic and efficient method for isolating and culturing term human placental trophoblasts was established in the study, expression and function of the CXCL12/CXCR4/CXCR7 axis in late pregnancy placental trophoblastic cells was analyzed, and the association between CXCL12/CXCR4/ CXCR7 axis with the apotosis mechanism of trophoblasts in preeclampsia. The goal of the study is explore these proteins'function in pathogenesis of preeclampsia, and provide new perspective for prevention and treatment in future. Part I The isolation and culture of term human placental trophoblasts Objective To establish an economic and efficient method for isolating and culturing human term placental cytotrophoblasts.Methods Term placentas were obtained from women having caesarean section with normal pregnancy, the tissue was digested repeatedly with Deoxyribonuc lease and trypsion, mesh screen filration and discontinuous density gradient centrifugation were used to isolate and purify the trophblast cells. Morphological changes of cells were observed under microscope and identified by immunohistochemical staining. CCK-8 proliferation assay and Trasswell assay were employed to analyze the viability and invasion ability of trophoblats in vitro.Result Trophoblast cells could be isolated and cultured successfully in vitro with this method. More than 107 trophobalst cells can be obtained at a time from approximately 50g tissues one time, cells began to stick wall after 3 hours and gradually began to merge into syncytiotrophoblasts after 24 hours. The proliferation and invasion capacity of trophobalst cells is low in vitro.Conclution This study improved the method for isolating and culturing term human placental trophoblasts, huge amount of purified cells can be obtained with more simplified operation in a rather short period of time.Part II The expression patterns of chemokine CXCL12/CXCR4/CXCR7 axis in trophoblast cells from human term placenta.Objective To investigate the expression pattern of CXCL12/CXCR7/CXCR4 in trophoblast cells isolated from normal human placenta, and to explore the biological function of CXCL12/CXCR4/CXCR7 in trophoblast cells.Methods Human term placentas were collected under the management of Ethics Committee department of Zhongnan Hospital of Wuhan University, and trophoblast cells isolation and cuture system as decribed in Part ?. The expression of CXCL12/CXCR4/CXCR7 were examined by Immunohistochemistry (IHC), Immunocytochemistry (ICC), Flow cytometry (FCM), and real-time PCR. The effect of rhCXCL12 and neutralizaing antibodies of CXCL12, CXCR4, and CXCR7 on proliferation of trophoblast cells were examined by CCK-8 assay. ELISA assay was carried out to detect CXCL12 secretion levels of trophoblast cells in viro.Results IHC and ICC disclosed specific staining for CXCL12, CXCR4, and CXCR7 protein in trophoblast cells. FCM and real-time PCR analyze the expression of CXCL12, CXCR4, and CXCR7 in trophoblast cells. CCK-8 assay shows rhCXCL12 has an positive effect on trophobalst cell survival, but the results indicated that rhCXCL12 increases the viability of trophoblast cells in a dose-independent manner, However this positive effect could be inhibited by neutralizing antibodies for CXCL12, CXCR4 or CXCR7. ELISA results showed that late pregnancy trophoblast cells in vitro culture could secrete CXCL12, but this secretion was time-independent. The concentration of CXCL12 was inhibited by neutralizing antibodies for CXCL12, CXCR4 or CXCR7.Conclusions CXCL12/CXCR4/CXCR7 were coexpressed in term placental trophoblast cells, which showed that the chemokine CXCL12/CXCR4/CXCR7 axis play some certain biological functions in late pregnancy process.Part ? The study of chemokine CXCL12/CXCR4/CXCR7 axis on the apotosis mechanism of trophoblasts in preeclampsiaObjective To explore the expression of CXCL12/CXCR4/CXCR7 in preeclampsia placental tissues and primary trophoblasts were to contribute to a better understanding of the association between these proteins and the pathogenesis of preeclampsia.Methods Samples from 11 mild preeclamptic patients,18 severe preeclamptic patients, and 21 normal pregnant women were collected. Morphological change of trophoblast cells that was observed by transmission electron microscope (TEM) and the apotosis rate of three groups tissues by TUNEL assay. The expression level of CXCL12 and its two receptors (CXCR4 and CXCR7) in these groups tissues were investigated by IHC. ICC, FCM, Real-time quantitative PCR (RT-qPCR) and Western blot were carried to detect the difference expression of CXCL12/CXCR4/CXCR7 between trophoblast cells isolated from normal and severe preeclamsia tissues. The effect of different treatment (the same as Part ?) on apoptosis of trophoblast cells measured with flow cytomety.Results TEM and TUNEL assay indicated a significant tendency of apoptosis in the preeclamptic placenta. IHC showed that expression level of three proteins was significantly lower in severe preeclamptic placentas compared with normal placentas (P<0.05), whereas no significant difference was found between mild preeclamptic and normal placentas (P<0.05). RT-qPCR and Western blot showed that both mRNA and protein expression level of CXCR4, CXCR7, and CXCL12 of trophoblasts were lower in the severe preeclampsia group than that in the normal group (P<0.05 for mRNA, P<0.01 for protein). The flow cytomety assay shows rhCXCL12 could reduce trophobalst cell apoptosis, and neutralizing antibodies for CXCL12, CXCR4 or CXCR7 may weaken the effection.Conclusions Our data revealed that the roles of CXCR4, CXCR7, and CXCL12 are associated with trophoblastic cells apoptosis and may be linked to the occurrence and development of preeelampsia. |
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