The Effect On Smad4of EVT Infected By HCMV Through TGF-β/Smad Pathway

Objectto observe the effect on Smad4of extravillous cytotrophoblast (EVT)infected byhuman cytomegalovirus(HCMV), and preliminary study the change of EVT’sproliferation and invasion function caused by HCMV.Methods1.we aseptically Collect villi that the healthy women in early pregnancy wasvoluntarily abortion, and use enzyme to digest and separate in order to culture EVT, Usingimmunocytochmical stain we detect cytokeratin7(ck7),vimentin(vim)and c-erbB-2toidentify source of cells.we apply the conventional method that we culture adherent cells toextravillous trophoblast cell line HTR-8and human embryonic lung fibroblast cell lineMRC-5.2.MRC5cells were cultured to amplify HCMV AD169, then we measure TCID50ofHCMV by a method of Reed-Muench.3.HCMVpp65antigen was detected to define if the primary EVT and HTR-8cells wasin the state of infection by immunofluorescence staining assay.4. MTT can measure the smallest viruses dose that can affect cell proliferation as afollow-tested best viral inoculation.5.We design two groups: control group(primary EVTand Virus group (primaryEVT+HCMV),and determine expression of Smad4proteins in every group by RT-PCR orimmunocytochemistry or Immunofluorescence staining and Western blot;6、To study invasion ability change of primary EVT cell infected HCMV,we useTranswell cell invasion assay in vitro.Results1.The consequence about cells’ source is that CK7and c-erbB-2antigens hadexpressed on95%cells and Vim antigen had scarcely expressed, Which suggested we hadobtained high purity primary EVT. 2.HCMV TCID50was10-4.21/0.1ml.3.there were great amount of red signal of HCMV pp65antigen in primary EVTwhichwas infected by HCMV, but the signal was not finded in cells uninfected;Immunofluorescence staining showed that HCMVpp65antigen fluorescent signals isdetected in the primary EVT cells.4.In the study, the best viral inoculation dose of30ul100TCID50HCMV/120ulmedium that1ul100TCID50HCMV join4ul high glucose DMEM medium. that30ul，40ul,50ul，70ul,100ul HCMV inhibited obviously proliferation of primary EVT at timepoint of48h after inoculated virus compared with control group(P<0.05), and each groupwas no significant difference (P>0.05).5.Real-time PCR shows that there was releated gene mRNA onTGF-β/Smad4expression in the virus group and the normal group. TGF-β1mRNA expression levels of thetwo groups were(1.001±0.052)和(1.296±0.114,) TβRI mRNA expression levels of the twogroups were（1.001±0.052）和（1.317±0.051）, Smad2mRNA expression levels of the twogroups were（1.000±0.033）和（1.380±0.064）, Smad4mRNA expression levels of the twogroups were （1.00±0.052）和（1.36±0.080）, Smad7mRNA expression levels of the twogroups were （1.317±0.051）和（1.001±0.052）, MMP2mRNA expression levels of the twogroups were（1.000±0.037）和（0.912±0.016）,MMP9mRNA expression level of the virusgroup was（1.000±0.037）和（0.909±0.025）, Smad1mRNA expression level of the virusgroup was(1.000±0.120和1.040±0.040), Smad5mRNA expression level of the virus groupwas(1.010±0.130和1.100±0.160), Smad6mRNA expression level of the virus groupwas(1.010±0.130和0.840±0.040).Compared with the control group, TGF-β1、TβRI、TβRII、Smad2、Smad3、Smad4mRNA expression levels increased significantly（P＜0.05）, Smad7、MMP2、MMP9mRNA expression levels increased significantly（P＜0.05）.6.Immunofluorescence staining shows that both the virus group and the normal groupcould express Smad4protein,and AOD of two group is (0.376±0.044)和(0.530±0.086),Compared with control group,expression of Smad4increased significantly inHCMV groups, P<0.05.7.Western Blot displays that both the virus group and the normal group could expressSmad4, GDR of two group is (0.518±0.053)和(0.884±0.093),and expression of Smad4increased significantly in HCMV groups, P<0.05. 8.Transwell cell invasion in vitro experiments suggest, EVT could cut throughMatrigel,the cell number of permeating Matrigel in control group and HCMV group of EVTwas （56.1±3.06）and（45.7±2.73) respectively(P<0.05),HCMV can inhibit the invasionfunction of primary EVT.Conclusion1.primary EVT can study HCMV infection as a experimental model cell.2.In vitro HCMV can decrease the growth and invasion of EVT cells by TGF-β/Smadand Smad4expression levels increased in the HCMV groups.3.Smad4is an important factor which takes part in the mechanism that abnormalpregnancy may be caused by HCMV infection. High expression of Smad4may decreaseinvasion of EVT, bringing about the occurrence of abnormal pregnancy abortion.