| BackgroudHuman cytomegalovirus, HCMV who is also known as human herpesvirus type5.It isone of the β subfamily in herpesvirus family, which is widely distributed in nature, humanpopulation is the only host. HCMV congenital infection is most common in pregnantwomen and intrauterine infection. Pregnancy after maternal infection HCMV, HCMV canpass through the placenta or reproductive tract retrograde spread to the embryo or fetalcause intrauterine infection. It can cause embryo stops growing, miscarriage, Stillb-irth, premature,intrauterine growth retardation and so on, After the intrauterine infectionchild was born they may have serious long-term consequences for example cognitiveretardation, mental retardation, sensorineural deafness. Until now the specific mechanismsand pathways of HCMV lead to intrauterine infection fetal abnormalities was stillunknown. HCMV infected the trophoblast cells of placental is considered as the first stepto start intrauterine infection. Poor proliferation and invasion of EVT was believed to beassociated with insufficient remodeling of the spiral arteries and dysfunction of placentalsubstance exchange, which was typical of the pathological changes of miscarriage,stillbirth, intrauterine growth restriction and neonatal brain developmental retardation.ObjectiveTo explore HCMV effect the function of regulate extravillous cytotrophoblasts byHippo-YAP pathway in vitro.Methods1. We collect pregnant villous tissue aseptically, An improved compoundenzymedigestion and gradient centrifugation method was used to get primary EVT.2. We detecte Cytokeratin7(CK7), vimentin (Vim) and c-erbB-2to identify source ofcells by immunocytochemical stain. 3. We choose the conventional method to culture MRC-5in the laboratory. TheHCMV AD169was passaged by cell culture and50%tissue culture infectious dose(TCID50) of HCMV was tested by the method of Reed-Muench.4. We use HCMV AD169to infect EVT. We use HCMV pp65antigen to detecte theinfection state of EVT by Immunofluorescence stain.5. We use MTT to test the effect on proliferation of EVT infected that can definite theoptimal virus inoculation for experiment.6. We divided them into control group and HCMV group, control group is normalEVT, HCMV group is EVT infected by HCMV.7. Real-Time PCR detected the expression of Mst1, Mst2, SAV, Lats1, Lats2, Mob1,YAP, TAZ, TEAD1~4mRNA in EVT of two groups.8. Immunofluorescence stain and western blot detected the expression of YAP proteinin EVT of two groups.9. The invasion of EVT was detected by cell invasion assay in vitro after they wereinfected by HCMV.Results1. The microscopic examination revealed that following cultures：the adherent cellsderived from pregnant villous tissue displaying triangle or polygon shape and patchyspreading growth.2. The identified result of cells’ source showed almost all of the cells isolatedexpressed CK7and c-erbB-2antigens, but scarcely expressed Vim antigen,It’s show thatwe had obtained high purity primary EVT.3. The HCMV AD169’s TCID50was10-4.15/0.1ml.4. A large quantity of red signal of HCMV pp65antigen in primary EVT which wasinfected by HCMV, but the signal was not detected in the primary EVT which was notinfected by HCMV.5. After HCMV infect EVT48h,30μl,40μl,50μl,70μl,100μl HCMV inhibited theproliferation of EVT obviously (P<0.05),30μl,40μl,50μl HCMV groups had notdifference significantly between them (P>0.05).we choose30μl100TCID50HCMV/120μlDMEM as the optimal virus inoculation for experiment.6. Mst1, Mst2, SAV, Lats1, Lats2, Mob1, YAP, TAZ, TEAD1, TEAD2, TEAD3,TEAD4mRNA are both express in EVT of the two groups, all the mRNA expression ratioin control group are1.000±0.014,1.000±0.036,1.000±0.025,1.001±0.047,1.000±0.016,1.001±0.067,1.002±0.067,1.000±0.017,1.001±0.053,1.001±0.050,1.000±0.039,1.009± 0.160, in HCMV group they are0.805±0.015,0.818±0.027,0.929±0.021,0.721±0.032,0.734±0.005,0.388±0.019,0.623±0.022,0.587±0.012,0.488±0.010,0.501±0.012,0.652±0.016,0.625±0.026, HCMV group compared with the control group the expression of themwere decreased significantly (P<0.05).7. Immunofluorescence detection that the levels of YAP protein were significantlylower in HCMV group than in control group. The average optical density of two groupswere0.271±0.024and0.159±0.017, compare with control group the expression of YAPprotein were decreased significantly in EVT of HCMV group,(P<0.05).8. Western blot detection that the levels of YAP protein were significantly lower inHCMV group than in control group. The gray date ratio of two groups were1.02±0.056and0.65±0.071, compare with control group the expression of YAP protein weredecreased significantly in EVT of HCMV group,(P<0.05).9. The result of cell invasion assay in vitro showed that that EVT could cut throughMatrigel， the cell number of permeating Matrigel in control group and HCMV group ofEVT was (55.7±2.95) and (47.9±3.21) respectively，compare with control group the cellnumber of permeating Matrigel were decreased significantly in EVT of HCMV group(P<0.05), which suggested HCMV had inhibited invasion of EVT.Conclusion1. In vitro HCMV can not only infect EVT but also inhibit the proliferation of them.EVT infected by HCMV can be used as the experimental model to research HCMVintrauterine infection.2. HCMV can decrease the mRNA expressed in Hippo-YAP pathway and YAPprotein in EVT, HCMV can be likely to inhibit invasion of EVT. |
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