| BackgroundHuman cytomegalovirus?human cytomegalovirus,HCMV?is human herpesvirus type 5,which is currently one of the most common virus herpes virus family?subfamily,ubiqu-itous in nature,humans are its only host,it is currently known to be causing active infection in pregnant women,the most common pathogen embryonic and fetal intrauterine infection[1].HCMV infection in pregnant women,can spread to the fetus,causing the embryo or fetal infection.But HCMV can cause fetal abnormalities specific mechanisms and pathways is not clear,it may be a route of transmission:vertical transmission placenta or genital tract retrograde spread,recently,the placenta and infection have become a hot topic,which is considered the main route of transmission.Pregnant women in early pregnancy can occur after infection HCMV miscarriage,premature birth,stillbirth and fetal growth restriction and other adverse pregnancy outcomes,even if reluctantly spent the fetus during pregnancy,after birth,is also facing a huge threat,showing microcephaly,cerebral edema,hepatitis,pneumonia,nervous system abnormalities,hearing impairment,mental retardation and other short and long term adverse outcomes[2].To date widely believed,HCMV infection placen-tal trophoblast cells is the first step to start intrauterine infection,and it is the most critical step in the outer hair cells trophoblasts?extravillous cytotroph-oblast,EVT?proliferation and invasion decreased,resulting in spiral artery recast bad placental material exchange dys-function,miscarriage,stillbirth,intrauterine growth restriction,brain retardation and oth-er abnormal pregnancy outcomes important pathophysiological mechanism.TGF-?/smadsignaling pathway TGF-?1 make decidualization of the regulation of trophoblast cell proliferation,invasion and adjust the structure of the placenta is formed and functioning,and smad4 is the only common-mediated smad,can after the formation of the receptor binding and phosphorylation smads heterologous compound,followed by the extracellular signals to the cell,acting on target cells exert their biological function.Therefore smad4 played a pivotal role throughout the TGF-?/smad signaling pathway EVT function.ObjectTo detect smad4 gene expression level by HCMV infection in vitro EVT,orientation increases,down smad4 genetic testing TGF-?/smad signaling pathway related gene expression levels,and explore HCMV by TGF-?/smad impact smad biological signaling pathway EVT invasion function.Methods1.We aseptically composite enzyme gradient centrifugation and cultured primary EVT,and identify the source and purity of the cells by immunocytochemistry.2.Were divided into normal control group?CTR?,smad4 interference group?I-smad4?,smad4 overexpression group?T-smad4?,HCMV infection?HCMV?.EVT respectively HCMV inoculated into cell 48 hours transfection siRNA smad4,DNAsmad424 hours,using indirect immunofluorescence staining,reverse transcription polymerase chain react-ion?RT-PCR?,Western blot?Western Blot?and other methods levels of expression of cell smad4 groups.3.We use transwell cell invasion assay in vitro to study invasion ability change of prim-ary EVT cell infected HCMV.Results1.About 95%of the EVT found CK7 and c-erbB-2,occasional Vim expression,the res-ults indicate that EVT isolated and cultured to a high purity,can be used for subsequent exp-eriments.2.RT-PCR results showed that four EVT messenger in both TGF-?RI,TGF-?RII,TGF-?1,TGF-?2,TGF-?3,MMP2,smad2,smad3,smad4,smad7 isogenic RNA?That mRNA?expression.Specific results are as follows:mRNA expression of TGF-?1 gene in four experiments were1.030±0.043,0.684±0.045,1.465±0.047,1.237±0.025;TGF-?2 gene expression relative to the amount of mRNA in the four groups were 1.015±0.210,0.354±0.116,0.546±0.070,0.527±0.025;TGF-?3 gene were 1.002±0.070,0.710±0.009,1.357±0.354,1.189±0.043 in the relative amount of mRNA expression of four grou-ps;TGF-?RI gene relative mRNA expression in four groups amounts were1.021±0.039,0.856±0.017,0.929±0.065,1.317±0.051,mRNA TGF-?RII relative gene expression in four groups in amounts of 1.034±0.073,0.87±0.0035,0.929±0.065,1.296±0.114;mRNA smad2 relative gene expression between the two groups in amounts of 1.000±0.033,0.806±0.0021,0.889±0.0457,1.380±0.064;mRNA smad3 gene expression levels in the two groups were relatively 1.005±0.13,0.983±0.0301,0.959±0.0477,1.34±0.065;mRNA of smad4 relative gene expression in four groups respectively was 1.00±0.052,0.150±0.0073,56.48±6.98,1.36±0.080;mRNA of smad7 gene in the relative expression amount of the four groups were 1.317±0.051,0.627±0.0026,0.857±0.0322,1.001±0.052;mRNA of MMP2 gene expression relative to the amount in four groups were 1.001±0.028,0.726±0.0188,0.926±0.00887,0.972±0.032.Compared with normal control group,HCMV infection group mRNA expression T?RI,T?RII,TGF-?1,TGF-?3,smad2,smad3,smad4 isogenic relatively increased,the differences were statistically significant?P<0.05?,mRNA expression levels of smad7,MMP2 and other gene is relatively decreased,the difference was statistically significant?P<0.05?.After smad4 regulated gene discovery,elevated TGF-?1,mRNA expression levels of TGF-?3 gene expression levels of mRNA expression of TGF-?2,TGF-?RI,TGF?RII,smad2,smad7 etc.significantly lower than normal group?P<0.05?;smad4 silence gene discovery,gene virus each group except smad3 no significant changes,the rest are reduced expression?P<0.05?.3.Immunofluorescence staining cells suggest that in four EVT both visible smad4expression,which in the normal control group,smad4 interference group,smad4overexpre-ssion group,HCMV infection group,the average optical density?AOD?were0.425±0.044,0.217±0.035,0.734±0.081,0.530±0.086.Compared with the normal group,HCMV infec-tion group EVT smad4 in protein expression levels were significantly increased,smad4 over than the other three groups,smad4 protein expression was significantly higher expression group,P<0.05,the difference was statistically significant,smad4 interference group smad4 protein The expression was significantly lower than the other three,P<0.05,differences were statistically significant.4.Western Blot results showed that the normal control group,smad4 interference gro-up,smad4 overexpression group,HCMV infection group were seen smad4 protein expre-ssion compared with normal control group,HCMV infection group smad4 protein increased,the difference was statistically significant?P<0.05?,after increases smad4smad4 gene expr-ession levels were significantly increased,the difference was statistically significant?P<0.05?;after down smad4 gene,smad4 protein levels decreased significantly?P<0.05?.5.In vitro cell invasion assay suggested that the normal control group,smad4interfere-nce group,smad4 overexpression group,HCMV infection group EVT can pass through Ma-trigel Matrigel normal control group transfected with HCMV group cell number was?57.3±2.16?,?46.2±2.18?a statistically significant difference,?P<0.05?,prompted HCMV inhibits EVT invasion vitality.Upregulated genes after smad4 down,EVT penetrating the cells were?36.1±2.14?,?65.3±3.21?months,compared with the normal group,the difference was statistically significant?P<0.05?,experimental study Tip invasion smad4 genes involved in the regulation of the EVT.Conclusion1.HCMV likely to inhibit the proliferation of cells and its primary EVT invasion func-tionality through TGF-?/smad signaling pathway.2.Smad4 gene in HCMV intrauterine infection can cause abnormal pregnancy outco-mes,which plays a role in the mechanism may be HCMV can damage normal structure of EVT cells,since a positive role in the regulation of TGF-?/smad signaling pathway,thereby promoting smad4 gene expression that EVT invasion insufficiency,and thus interfere with embryo implantation,resulting in various abnormal pregnancy outcome. |
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