| As an important organ responsible for the exchange of substances between mother and fetus,the placenta is crucial to the growth and development of the fetus during pregnancy.Its dysfunction will lead to fetal growth restriction,preeclampsia and other diseases,and even cause fetal death.The endocrine function of trophoblast is one of the important functions of placenta and plays an indispensable role in the development of fetus.However,the mechanism of regulating the endocrine capacity of trophoblast cells is still unclear.As A diazepam binding inhibitor(DBI),also known as acyl-coa-binding Protein(ACBP),DBI is highly enriched and expressed in steroid-generating cells.Numerous studies have shown that DBI is involved in the regulation of steroid production in a variety of cell types.However,it is not clear whether it is involved in the regulation of placental trophoblast DE novo anabolic steroids.In this study,the epidemiological characteristics of patients with spontaneous abortion were analyzed,and the expression and localization of DBI in the spontaneous abortion group and normal control group were determined by immunohistochemistry,immunofluorescence,RT-qPCR,western blot and other experiments using human pregnancy villi tissue and trophoblast cell line as models.Then,after the expression in trophoblast cells was knocked down with siRNA,the expression level of hormone synthase was detected,and the effect of DBI expression on cell proliferation was detected.The following results were obtained: 1.Comparison of epidemiological characteristics of patients with spontaneous abortionThe patients with spontaneous abortion were divided into the elderly group by age,and the basic data and pregnancy history of the two groups were analyzed.The age of first marriage of the elderly group was higher than that of the age group,and the number of pregnancies,term births and induced abortions was higher than that of the age group.2.Comparison of DBI in spontaneous abortion and normal pregnancy villi tissue.Collect spontaneous abortion cases fluffy villi tissue and normal villi tissue as control group,Western blot was used to compare the expression difference of DBI in the villi tissues of the two groups.Immunohistochemistry was used to compare the expression position of DBI in the two groups.Immunohistochemical results showed no significant difference in the expression and location of DBI in the villi tissues of the two groups,while Western blot results showed that DBI expression in the spontaneous abortion group was lower than that in the control group.3.Expression pattern of DBI in trophoblast cells.Immunohistochemistry and immunofluorescence were used to analyze the expression location of DBI in the villi tissue,and the expression of DBI in the cell model was detected by RT-qPCR and western blot.The results showed that DBI was expressed in the cytotrophoblastic(CTB)layer,syncytiotrophoblastic(STB)layer and extracellular trophoblastic cells(EVTs)of the villi of early pregnancy,and the expression of DBI was induced by Forskolin.4.Effect of DBI expression on hormone synthesis.Cell immunofluorescence,RT-qPCR,western blot and other techniques were used to investigate the effect of differential expression of DBI on steroid hormone synthesis in BeWo cell line.The results showed that: in the induced zygocytosis group,the expression of DBI and shear enzymes increased,and the knockdown of DBI reduced the expression of steroid hormone shear enzymes.To explore the effect of DBI on steroid secretion,BeWo cells were treated in the following groups: control siNC group,siDBI knockout DBI group,FSK-induced zygocytosis group,and siDBI-FSK combined treatment group.RT-qPCR was used to detect the expressions of DBI,?-hCG and steroid hormone shear enzymes(CYP11A1,HSD3?1,CYP19A1).The results showed that DBI significantly reduced the expression of steroid ratelimiting enzymes,and Forskolin induced the expression of DBI shear enzymes in BeWo.5.DBI affects the proliferation capacity of trophoblast cells.Estrogen and progesterone are known to regulate autophagy in mouse uterine stromal cells,and abnormal progesterone signaling leads to continued proliferation of uterine epithelium.We would like to know whether the effect of steroid hormone produced by DBI will affect the proliferation of trophoblasts.The effect of DBI on the proliferation ability of BeWo cells was observed by EdU experiment.The results showed that the positive(green fluorescence)ratio was significantly lower in the DBI knockdown group and the FSK-induced group than in the control group.The proliferation ability of BeWo cells was also reduced when DBI expression was knocked down.Based on the above results,we believe that DBI is widely expressed in trophoblast cells and affects the DE novo anabolic steroid hormone of trophoblast cells by affecting the expression of steroid hormone synthase and the proliferation capacity of trophoblast cells.DBI may affect the expression of trophoblast anabolic hormone gene by regulating P450 scc,which may be related to placenta-related diseases during pregnancy.It provides new ideas for early screening and treatment of spontaneous abortion. |
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