Construction Of Placental-specific CD81 Transgenic Mice And Identification Of Its PE Phenotype

Background Preeclampsia is a pregnancy-specific syndrome which is the leading cause of maternal and fetal mortality and morbidity in both developed and developing countries.Although until now its pathogenesis remains unknown,It is well believed that PE is initated by abnormal placentation in the early stage of pregnancy which is due to the impaired invading function of cytotrophoblasts(CTBs),and followed by systemic activation of endothelial cells of the maternal small arterioles in the late second or third trimester of pregnancy.However,it is still unclear how the invasion of CTBs is regulated during the process of placenta formation.Our previous study showed that CD81,a member of the tetraspanin superfamily,was preferentially expressed in normal first trimester placental CTBs and progressively downregulated with gestation advance.While in patients with early-onset severe PE(sPE),CD81 expression was significantly up-regulated in syncytiotrophoblasts(STBs),CTBs and the cells in the villous core as well as in the materal sera which indicates the important role of CD81 in the pathogenesis of PE.To make sure the above finding in vivo and further explore the mechanism,Here,we use syncytin promter to establish a placenta-specific CD81 high expression transgenic mice model and further evaluate its phenotype of PE,as well as the molecular pathological changes.Methods The recombinant plasmid of pGL3-syncytin1 promoter-CD81 was constructed and the transcriptional activity of this plasmid in the trophoblast(HTR8/SVneo and BeWo cells)and non trophoblastic cells(IshiKawa,COS7,HUVEC and HGC-27 cells)was detected by real-time fluorescence quantitative PCR(qRT-PCR).Then,the plasmid was integrated into the mouse genome by microinjection to construct the syncytinl promoter-CD81 transgenic founder mice.The founder mice were hybridized with C57BL/6 wild type mice,and the positive offspring were identified by PCR.The offspring information was analysed to identify the genetic stability of transgenic mice and the genetic background purification was also in progress.At the same time,qRT-PCR,Weatern blot and immunohistochemical staining were used to identify the expression level of CD81 in the transgenic mice.Finally,the transgenic female and male mice were mated with the C57BL/6 wild type mice respectively.The tail sleeve method was used to detect the blood pressure,and the statistical analysis of the fetus number and weight,the HE staining of the kidney,the HE and the immunohistochemical staining of the placenta were uesd to observe whether the parental-and maternal-derived high expression of CD81 could cause the PE symptoms.Results The pGL3-syncytinl promoter-CD81 recombinant plasmid was activated in HTR8/SVneo and BeWo cells with the CD81 expression increased by 10.86+0.40 times(P=0.0001)and 21.83+7.64 times(P=0.0344),but no significant changes were observed in IshiKawa cells,COS7 cells and HUVEC cells.However,in HGC-27 cells,the CD81 expression increased by 9.10+0.63 times(P<0.0001).Syncytinl promoter-CD81 transgenic founder mice and their offspring could identify the transferred gene,and the offspring positive mice accounted for 44.2%.At transcriptional level,the placental CD81 mRNA in the transgenic offspring was significantly higher than that of the wild type rats,among which the parental-derived transgenic mice increased by 16.23+3.793 times(P=0.0011),the maternal-derived transgenic mice increased by 3.61+0.3364 times(P<0.0001),while the CD81 mRNA did not rise in the heart,liver,spleen,lung,artery,muscle and other organs.At the protein level,WB detection showed the increase of protein CD81 in the placentas of CD81 transgenic mice,and CD81 immunohistochemical staining of placentas showed more CD81 expression in the transgenic mice which distributed alomst in the placental labyrinth layer.The blood pressure of pregnant mice in transgenic groups began to rise from GD9,and increased gradually until GD17.The glomerular swelling of pregnant rats and low-weight of fetus were also observed(0.812+0.010g in the control group,0.766+0.0168,P<0.05,0.799 + 0.012g,P>0.05 in the maternal-derived transgenic group).As for the placental spiral artery staining,in CD81 transgenic mice,there was CK-7 staining negative and alpha-SMA staining positive in the placental mesangial triangle area,which was opposite to that in control group.Conclusion The pGL3-syncytinl promoter-CD81 recombinant plasmid can activate CD81 transcription specificly in the trophoblast cell lines.And the syncytinl promoter-CD81 transgenic mice can be genetically stable and realized the placental-specific high expression of CD81.Both parental and maternal-derived CD81 high expression in fetal placentas can lead to PE symptoms such as hypertension,renal injury,and fetal growth restriction,which may result from the deficiency of the placental spiral artery remodeling.