| Objective Preeclampsia is a characteristic diseases during pregnancy,which is the main cause of maternal and fetal morbidity and mortality.High blood pressure and proteinuria are main clinical manifestations of this disease which usually happened after 20 weeks of gestation involves systemic disease.The incidence of pregnant women 5% to 8%.A large number of hypotheses and theories have been established to explain the etiology of preeclampsia,such as abnormal immune response,oxidative stress,abnormal placental implantation and placental function,and genetic factors.Foreign scholars had put forward the "two-stage theory" of the pathogenesis of preeclampsia.The score content includes: at the first stage of early pregnancy,including immune,genetic and other factors,such as endothelial cell disorder can lead to disorder of "vascular recasting" in uterine spiral artery,decreased the invasiveness of nourish cells,which cause "shallow implantation" increase the resistance of uterine artery lead to the decline of placental function.At the second stage of later pregnancy,endothelial cell damage triggered by oxidative stress response in local placenta caused by ischemia hypoxia,released a large number of inflammatory factors,forming the inflammatory cascade effect and excessive inflammations related to various clinical symptoms of preeclampsia and eclampsia.Although a lot of research on the etiology and pathogenesis had been done at home and abroad,there is no single theory that can be satisfied and fully explained.Therefore,the study on the pathogenesis of preeclampsia is still a challenge for obstetricians.Chemokines are polypeptides containing four conservative cysteine residues and chemotactic chemotaxis.According to the number of other amino acids contained in the first and second cysteine,it can be divided into CXC,CC,C and C3XC4 subgroups.Chemokines play different roles by binding to receptors of different cell types.In the n-terminal area(CXC,CC,CX3 C and C),chemokines can be divided into four main groups.The main role of chemokines is to attract immune cells to the immune response and participate in immunoregulation and pathological reactions.Chemokines play an important physiological and pathological effect in the body,and the development of diseases such as inflammation,tumor and autoimmune diseases has the abnormality of chemokines or their receptor expression.Preeclampsia has been reported to be closely related to the occurrence of hypertension,diabetes,and congenital heart disease in offspring.The use of ultrasound Doppler to measure the abnormality of the cardiac axis has the characteristics of high sensitivity and specificity for screening the fetal heart.However,it is unclear whether the abnormalities of the fetal heart in preeclampsia have abnormal expression of certain chemokines.C-X-C motif chemokine ligand 9 also called MIG,CXCL9 genes located on chromosome 4 q21,belong to induced by interferon gamma CXC chemokines family,mainly distributed in Th1,B cells and NK cells,mediated and regulating immune and inflammatory reaction.CXCL9 can’t be detected in most of the tissues in normal physiological condition,but it can be induced by interferon gamma when infection,injury or immune inflammatory reaction occurs.CXCL9 receptors are cxc-subfamily CXCR3.CXCR3 is also a receptor for chemokine CXCL10 and CXCL11.The chemotaxis of CXCL9 is accomplished mainly through the mediating of CXCR3.In our previous study,identification of differential gene expression profiles in placentas from preeclampsia pregnancies versus normal pregnancies by DNA microarrays.The results showed that CXCL9 m RNA were down regulated in preeclampsia placentas compared with normal placentas.It is not clear whether the localized expression and quantitative expression of proteins in trophoblast cells are also different.The purpose of this study is to investigate whether the localization and quantitative expression of CXCL9 protein in normal pregnancy and preeclampsia placental trophoblasts were different on the basis of previous studies and to investigate the proliferation,migration,invasion,and Akt signaling pathway.And further explore CXCL9 to participate in the occurrence and development mechanism of action of preeclampsia.Medthods Part One: Case screening and differential expression of CXCL9 protein in placental trophoblast cells of preeclampsia.From September 2015 to August 2016,30 patients with preeclampsia in the First Hospital of China Medical University were recruited.30 cases of pregnant women with healthy pregnancy were control.This study was approved by the ethics committee of the First Hospital of China Medical University.All subjects signed informed consent,and age,height,weight and other general conditions match in both groups with regular menstruation,and normal pregnancy process,without any complications and comorbidities(with the exception of preeclampsia),and without adverse pregnancy history,without previous essential hypertension,diabetes,kidney disease,cancer,etc,,no tobacco,alcohol and other addictions;The included pregnant women were in their first pregnancy,with single fetus.The gestational weeks was carefully calculated and checked by the date of the last menstrual period and early pregnancy ultrasound.The expression of CXCL9 in the placenta of preeclampsia was detected by immunohistochemistry.Western blot and Real-time PCR were used to detect the protein and m RNA expression of CXCL9 in preeclampsia placenta.Part Two: Effects of CXCL9 on proliferation,migration and invasion of trophoblast HTR-8/SVneo.Trophoblast cells HTR-8/SVneo was cultured in vitro,the CXCL9 gene was knocked out by sh RNA lentivirus infection.The CCK-8 assay was used to detect the proliferation of trophoblast HTR-8/SVneo under different intervention conditions;transwell assay was used to detect the CXCL9 gene knockout,changes in migration and invasive ability of trophoblast HTR-8/SVneo.Part Three: Effect of CXCL9 on the invasive mechanism of trophoblast HTR-8/SVneo.Trophoblast cell HTR-8/SVneo was cultured in vitro,CXCL9 gene was knocked out by sh RNA lentivirus infection.Western blot was used to detect the expression changes of MMP-2 and MMP-9 in trophoblast cells before and after knockout of CXCL9 gene;application of specific Akt inhibitors to detect the effect of CXCL9 on downstream Akt signaling pathways.Results Part One: We used immunohistochemistry,Western blot and Real-time PCR to detect the location and expression of CXCL9 in preeclampsia placental trophoblasts and normal pregnancy placental trophoblasts.The results showed that CXCL9 was positively expressed in the nucleus and cytoplasm of placental trophoblasts in normal pregnancy.In the preeclampsia patients,the expression of placental trophoblast nucleus and cytoplasm was weakened and some trophoblasts were lacking in expression.Statistically significant differences in the average density of placental trophoblasts in preeclampsia were significantly reduced.Western Blot and Real-time PCR showed that the protein and m RNA expression of CXCL9 in placenta of preeclampsia was significantly lower than that in normal placenta.The difference was statistically significant.It is suggested that the localization and quantification of CXCL9 protein and m RNA expression may be closely related to the function of placental trophoblast cells.Part Two: CCK-8 cell proliferation assay was used to examine the effect of CXCL9 on the proliferation of trophoblast cells HTR-8/SVneo.The proliferation ability of trophoblast HTR-8/SVneo after infection with sh RNA lentivirus was significantly reduced.Transwell assay was used to detect the ability of cell migration and invasion.At the same time,the ability of migration and invasion of trophoblast HTR-8/SVneo infected by sh RNA lentivirus was also significantly reduced..Part Three: Compared with the negative control group,after CXCL9 gene knockdown,the protein expression of MMP-2 and MMP-9 in trophoblast cells was significantly decreased,phosphorylated Akt(p-Akt)was significantly down-regulated,and phosphorylated ERK(p-ERK)levels were no change.In trophoblasts without CXCL9 knockout,MMP-2 and MMP-9 protein levels were significantly down-regulated after used with Akt inhibitor MK-2206.Conclusions 1.The protein expression of CXCL9 in placenta of preeclampsia was significantly lower than that of placenta in normal pregnancy,indicating that the low expression of CXCL9 may be the main factor of placental trophoblastopathy in preeclampsia..2.CXCL9 may play an important role in promoting the proliferation,migration and invasion of trophoblast cells HTR-8/SVneo.3.The up-regulation of MMP-2 and MMP-9 by CXCL9 may be achieved through regulation of the Akt pathway,but may not be directly related to the ERK signaling pathway.. |
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