Expression Of TNFAIP8 In Early Pregnancy Trophocytes And Effect Of TNF-AIP8 SiRNA On Apoptosis And Invasiveness In Trophocytes

ObjectiveThis study use early pregnancy villous trophocytes to process the vitro experiments simulating the placenta formation of the initial environment,clear whether TNFAIP8 expressed in early pregnancy trophocytes.By adenovirus transfection technique silence TNFAIP8 gene of early pregnancy villous trophocytes,and by flow cytometry,transwell,scratch experiment to explore in the cells of gene silencing,the influence of cells'apoptosis,attacks and migration ability,for the clinical management of preeclampsia provide new targets for intervention.TNF-AIP8 involved in regulating and controlling apoptosis of cells,and found it plays an important role in regulating cell signal transduction,tumor cell proliferation,invasion,metastasis and inflammatory reaction.In early pregnancy,the villous extral trophocytes were involved in the reconstruction of the uterine spiral artery,once the attack ability be impaired can lead to a spiral artery remodeling disorder,leading to"placenta implantation",the occurrence of preeclampsia.In the early stage of the study,using the global-wide human genome DNA methylation chip analysis placenta tissue of preeclampsia and normal pregnant women,found TNFAIP8 low methylation on preeclampsia placenta,it is mainly expressed in trophoblast cells,and the expression of both TNFAIP8 has difference^[1].Therefore,TNFAIP8 protein may participate in the regulation of the number and invasion capacity of trophoblast cells and interfere with the process of the uterine spiral artery remodeling,leading to the implantation of the placenta,and participate in pre-eclampsia.Methods1.Using immunohistochemical method verify normal early pregnant trophocytes to judge TNF-AIP8 positive expression.2.Take the villous tissue and peripheral blood,through Westernblot check the expression of TNFAIP8 protein was correlated.3.By the Shanghai sangong compose siRNA pTrack-CMV plasmid and empty plasmid,and transfected the 293 cell to obtain the virus particles,its transfect HTR8/SVneo cells,and test transfection effect by RT-PCR and Western blot.The cells can be divided into 3 groups:TNFAIP8 silent group?with TNFAIP8-siRNA in virus infection?,blank control group?not infected HTR8/SVneo cells?and the negative control group?with Scr-siRNA in the virus?.Compare the invasion and migration ability of three groups by Transwell little chamber experiment and cell scratch experimental.And using flow cytometry instrument studies three groups of cells apoptosis rate.Results1.TNF-AIP8 in the cytoplasm of early pregnancy trophocytes positive expression.and the TNFAIP8 protein content was positively correlated between trophocytes andperipheral blood lymphocytes.2.Transfect the virus particals which producted in the pTrack-CMV contain siRNA?scrRNA to HTR8/SVneo cells,then use RT-PCR and Western blot to test TNFAIP8gene silencing effect.TNFAIP8 silent group compared control groups,the protein expression and mRNA of TNFAIP8 were significantly reduced?P<0.05?.Successful build the TNFAIP8 silent group,blank control group and the negative control group.The three groups of cells were tested for Transwell?cell scratch test and apoptosis test.The results showed the at the cell number through the pore in TNFAIP8 silent cell group higher than the other two groups and the difference was statistically significant difference?P=0.000?.The cell scratch test confirmed that TNFAIP8 silencing cell group was wide in the width of the blank control group and the negative group,and the difference was statistically significant?P=0.000?.By FCM method,compare the apoptosis rate of the three groups of cells after 36h,the apoptosis rate of TNFAIP8 silent cell group was higher than that of the other two groups,and the difference was statistically significant?P<0.05?.Conclusion1.TNF-AIP8 in normal early pregnancy trophocytes positive expression,and TNF-AIP8 lower expression influence trophocytes invasion and migration ability,increase the rate of cell apoptosis.2.TNF-AIP8 protein content in early pregnancy peripheral blood lymphocytes and villi tissue are positive correlation.3.TNFAIP8 is expected to be for clinical early diagnosis of preeclampsia prediction.