| BackgroundRecurrent spontaneous abortion(RSA)refers to two or more spontaneous abortions with the same spouse in early pregnancy.It is estimated that RSA occurs in 2%-5%of women of childbearing age.It is a common pregnancy complication and has become a major social health problem in my country.The etiology of RSA is complex.The currently identified pathogenic factors include genetic factors,endocrine factors,and immune factors.But still about 50%of patients have unknown etiology,which is called unexplained recurrent spontaneous abortion(URSA).Most of URSA occurs in the first trimester of pregnancy,and there is no treatment for the cause,which makes patients feel very distressed and helpless and has seriously threatened women’s reproductive health.In 2015,since the full implementation of the second-child birth policy in my country,the number of pregnant women of advanced age has gradually increased,and the incidence of URSA has shown a significant increase.URSA not only seriously affects the reproductive health of women of childbearing age,but also poses a huge threat to patients,their families and society.Therefore,it has important clinical value and research significance to further explain the pathogenesis of URSA,reveal its intrinsic molecular mechanism,and explore its diagnosis and treatment methods.The placenta is a multifunctional organ that is essential for fetal development and survival.Placental dysplasia can lead to a variety of pregnancy-related diseases,such as abortion,fetal growth restriction and preeclampsia.Normal development of the placenta is closely related to changes in the partial pressure of oxygen within the placenta,which is reported to develop initially in a hypoxic environment containing 1-2%oxygen until the 10th week of pregnancy.Trophoblasts are cells with special functions in the placenta.In order to ensure the normal progress of pregnancy,trophoblasts regulate their biological characteristics such as differentiation,proliferation,invasion and migration under the combined action of various mechanisms.Cell migration is an important biological process that contributes to the establishment of the placenta during pregnancy.The hypoxic environment of the placenta in early pregnancy provides optimal conditions for trophoblast invasion and placental formation.This change in partial pressure of oxygen can affect the proliferation and invasion ability of trophoblast cells,which in turn affects the development of the placenta.hypoxia inducible factor-1α(HIF-1α)is reported to be highly expressed in placental trophoblast cells at 5-8 weeks of gestation,but significantly decreased after 10-12 weeks with increased oxygen levels.It is found that hypoxia can promote the proliferation and invasion of trophoblast cells.Therefore,hypoxia is essential for early blastocyst implantation and trophoblast cell proliferation,differentiation and migration.Hypoxia is one of the characteristics of most solid tumors,and hypoxia microenvironment also becomes an important feature of solid tumor microenvironment.Differentiated Chondrocyte gene 1(DEC1)is a member of the basic helix-loop-helix(bHLH)transcription factor family.It plays an important role in various physiological functions such as stress response,circadian rhythm and cell proliferation and differentiation by regulating the expression of downstream target genes.Studies have shown that DEC1,as a hypoxia regulating gene,is a marker of tumor hypoxia and plays an important role in the occurrence and development of tumors.In addition,DEC1 expression is closely related to hypoxia inducible factor-1α(HIF-1α)in many solid tumors such as liver cancer,gastric cancer and pancreatic cancer.Early placentas,like tumors,develop in a hypoxic microenvironment.The phenotypes of the two entities may share molecular pathways,an important feature of which is the microenvironment in which they develop,in particular the availability of oxygen.Previous studies have found that DEC1 is involved in embryo implantation and pregnancy as a rhythm gene,but the expression and mutual regulation of HIF-1α and DEC1 in early pregnancy have not been reported.Aims1.Detect the expression and distribution of HIF-la and DEC1 in the villi of normal abortion patients and URSA patients,and analyze the correlation between the two expressions.2.To detect the expression changes of DEC1 and HIF-la in human trophoblast cell line HTR-8/SVneo under normoxia and hypoxia,and to explore the effect of hypoxia on the expression of DEC 1.3.Obtain a trophoblast cell line with stable and low expression of DEC 1,which provides a good cell model for the subsequent study of the mechanism of DEC1.4.At the cellular level,the effects of hypoxia on DEC1 expression,cell proliferation,migration and invasion were further explored.5.To establish a pregnant rat model to detect the mutual regulatory relationship between HIF-1α and DEC1 and its effect on the early placental development of pregnant rats.Methods1.Collect the villus tissues of patients with normal abortion and URSA at 6-10 weeks,detect the expression levels of HIF-la and DEC1 by RT-PCR and immunohistochemistry respectively,and analyze the correlation between the two expressions.2.The trophoblast cell line HTR-8/SVneo was cultured in normoxia and hypoxia.RTPCR and Western Blot were used to detect the expressions of HIF-la and DEC1 in the two groups of cells respectively;CCK-8,scratch and Transwell migration and invasion assays were used to detect the proliferation,migration and invasion of the two groups of cells.3.Construct DEC1 interference(DEC1 shRNA)and negative control(Nc shRNA)vectors,and screen to obtain cell lines with stable and low expression of DEC 1;RT-PCR and Western Blot were used to verify the validity of the vectors.4.The above DEC1 interference and control cell lines were cultured in normoxia and hypoxia respectively;CCK-8,scratch and Transwell migration and invasion assays were used to detect the proliferation,migration and invasion of trophoblast cell lines in each group.5.The protein inhibitor 2ME2 of HIF-1α was added to HTR-8/SVneo cells,and the expression changes of DEC 1 were detected by RT-PCR and Western Blot respectively,which further explained the expression relationship between HIF-1α and DEC1.6.The pregnant rat model was established,and 2ME2,a protein inhibitor of HIF-1α,was administered intraperitoneally.At the same time,the control group was intraperitoneally injected with the same amount of DMSO+corn oil as the 2ME2 intervention group.Western Blot and RT-PCR were used to detect the expressions of HIF-la and DEC1,respectively;The developmental process of placental fetuses in the two groups of pregnant rats was observed;Western Blot and RT-PCR methods were used to detect the invasion of placental villous trophoblast cells in the two groups of pregnant rats.Results1.Compared with the control group,the expression of HIF-1α and DEC1 in URSA group was significantly down-regulated,and there was a significant correlation between HIF1α and DEC1 expression.2.Hypoxia induces the expression of HIF-1α and DEC1,and promotes the proliferation,migration and invasion of trophoblast cells.3.We obtained a stable trophoblast cell line with low expression of DEC1.4.DEC1 promotes the proliferation,migration and invasion of trophoblast cells.5.DEC1 expression is regulated by HIF-1α under hypoxia.6.Animal experiments showed that DEC1 expression was regulated by HIF-1α.At the same time,2ME2 could affect the development of rat embryos and reduce the invasion of trophoblast cells.Conclusion1.There was a significant correlation between HIF-1 α and DEC1 expression in the villous tissues of patients with early pregnancy.2.In HTR-8/SVneo cells,hypoxia can induce HIF-1 α and DEC1 expression,and promote the proliferation,migration and invasion of trophoblast cells.3.DEC1 promotes the proliferation,migration and invasion of trophoblast cell lines through HIF-1 a pathway under hypoxia.4.Combined with in vitro and in vivo experiments,we believe that abnormal expression of HIF-1α/DEC1 signal may be closely related to the occurrence and development of URSA. |
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