| ?Background?Successful implantation of the human embryo depends on the delicate chorus between the differentiation of endometrial stromal cells into decidual cells and the ectoderm of the embryo into the trophoblast cells.Trophoblast cells share several common features with malignant cells(highly proliferation and highly invasion)to penetrate the endometrial stroma for the functional placentation,in particular during the first trimester of the pregnancy.Under normal physiological conditions,this process is precisely regulated by various cytokines/chemokines,growth factors and hormones in an autocrine or paracrine manner at the maternal-fetal interface.These factors control trophoblasts proliferation,invasion and apoptosis.Dysfunctional interation of trophoblasts with the mother-derived cells disturbs the physiological functions of multiple cells at the materno-fetal interface,which is linked with gestational complications,such as pre-eclampsia,intrauterine growth restriction,miscarriage and so on.Chemokines are a superfamily of chemotactic cytokines of low-molecular-weight(7-15 k Da),which contain 70 to 90 amino acids and play a variety of physiological roles through binding to the corresponding G-protein-coupled receptors.48 chemokines and 19 different chemokine receptors have been identified in humans up to now.We have previously demonstrated that conditioned medium from human decidual stromal cell(DSCM)produce a concentration-dependent effect on human trophoblasts(B6Tert)invasion,which addressed that 5%DSCM enhanced the trophoblasts invasion,while 20%DSCM inhibited its invasion.To further study the exact mechanism,we screened the protein-microassay to analyze the different expression profile cytokines in treated trophoblasts.We found the expression of CCL28 was controversial,which meaned the chemokine was significantly increased in both 5%DSCM and 20%DSCM treatment.Besides,the recent studies have found that CCL28 could promote the invasion of a wide range of cancer cells and CCL28 could induce the apoptosis of decidual stromal cells in human spontaneous abortion.Therefore,we hypothesize that CCL28 might play a role in early pregnancy on trophoblasts proliferation,apoptosis and invasion functions.?Objective?1.To explore the impcact of different concentration of DSCM(5%,20%,50%,100%)on proliferation,apoptosis and invasion of trophoblasts(HTR-8/Svneo cell line)in vitro.2.To investigate whether DSCs from the first trimester influence the m RNA expression and the protein secretion of CCL28 in trophoblasts.3.To explore the potential effect of CCL28/CCR3/CCR10 axis on the proliferation,apoptosis and invasion of trophoblasts and the related molecular mechanism of these biological functions.?Methods?1.Isolution,culturing and indentifying the purity of DSCs from healthy women of early pregnancy to prepare different concentration of DSCM(5%,20%,50%,100%).MTT,Annexin V-FITC/PI,Transwell were used to investigate the impcat of DSCM on trophoblasts proliferation,apoptosis and invasion.2.Using RT-PCR to detect the m RNA expression of CCL28 and its receptor CCR3 and CCR10 of trophoblasts treated with different concentrations of DSCM;Protein secretion of CCL28 in the supernatant of DSCs,HTR-8/Svneo and their co-culture was detected by enzyme linked immunosorbent assay(ELISA).3.The effect of CCL28 on trophoblasts proliferation,apoptosis and invasion were assessed by the MTT assay,Annexin V-FITC/PI staining and Transwell chamber;MMP-2 and MMP-9 activity of trophoblasts was treated with CCL28 in the invasion assay was analyzed by RT-PCR and gelatin zymography to further study the related molecular mechanism on the invasion function.?Results?1.DSCs could isolate and culture successfully in vitro and we could obtain a purity over 90% of DSCs;With the concentration of DSCM increased,the proliferation of trophoblasts been enhanced and the 100%DSCM induced the largest amount of proliferation compared with the control group(P<0.05);DSCM inhibited the early apoptosis,late apoptosis and total apoptosis of the trophoblats in a dose-dependent manner with significant difference was found between 100%DSCM and control group(P<0.05);And the effect of DSCM on trophoblasts invasion yielded the similar results,significant difference of cell invasion were found both in 50%DSCM,100%DSCM with control group(P<0.05).2.The m RNA transcription of CCL28 was elevated in the trophoblasts which was treated with DSCM(P<0.05),but neither its receptor CCR3 nor CCR10 was changed(P>0.05);The DSCs and trophoblasts could secrete CLL28 inceased with time,and their co-culthure could promote the secreted of CCL28 markedly(P<0.01).3.CCL28 did not affect the proliferation(P>0.05)or apoptosis of trophoblasts(P>0.05)but promoted its invasion(P<0.01)in a proper concentration through binding with its CCR10 receptotor(P<0.01)mainly.And CCL28 promoted cell invasion by enhancing MMP-2 activity(P<0.01).?Conclusion? Decidual microenviroment could enhance trophoblasts proliferation,invasion and inhibite its apoptosis;And the first-trimester DSCs could promote the trophoblasts to secrete CCL28,which may play an impotant role in early pregnancy for trophoblastic invasion by binding with receptor CCR10 through altering the expression of MMP-2. |
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