The present study examined von Willebrand factor (vWF) levels and ADAMTS13 activity in pregnant and severe preeclamptic women in order to shed light on the prothrombotic state in severe preeclampsia.Thirty healthy women of childbearing age,22 second trimester pregnant women,30 third trimester pregnant women and 10 severe preeclamptic patients were recruited in this study.ADAMTS13 activity was determined by the FRETS-vWF73 assay and vWF antigen (vWF:Ag) levels by an enzyme-linked immunosorbent assay.The results showed that there were statistically significant differences in plasma vWF antigen levels between the severe preeclamptic and third trimester pregnant women,between third and second trimester pregnant women (P<0.05).The third trimester pregnant women had significantly lower plasma ADAMTS13 activity than second trimester pregnant women (P<0.05).Nevertheless,no significant differences in plasma ADAMTS13 activity were found between severe preeclamptic patients and the third trimester pregnant women (P>0.05).In conclusion,plasma ADAMTS13 activity is normal in severe preeclampsia despite the increased vWF:Ag levels.Prothrombotic state is involved in the pathogenesis of severe preeclampsia,as a result of endothelial injury.
The effect of human cytomegalovirus(HCMV)on invasive capability of early pregnant extravillous cytotrophoblasts(EVTs)was investigated in vitro.Primary EVTs were obtained by com-plex phosphoesterasum digestion and gradient centrifugation from villous tissue aseptically taken from healthy pregnant women.Cytokeratin7(CK7),vimentin(Vim)and c-erbB-2 were immunocy-tochemically detected to identify source of cells,and HCMVpp65 antigen was assayed to determine the infection state of primary EVTs by immunocytochemical staining.The EVTs were divided into two groups:control group and HCMV group,and the expression of c-erbB-2,matrix metalloproteinase-2(MMP-2)and MMP-9 proteins was detected in two groups by immunocytochemistry and Western blotting.Enzymic activity changes of MMP-2 and MMP-9 were tested by gelatin zymography in primary EVTs infected with HCMV.The invasion of primary EVTs was detected by cell invasion assay in vitro after they were infected by HCMV.The cell source identification showed that the cells obtained were highly-pure primary EVTs,and primary EVTs could be infected by HCMV.Primary EVTs could express c-erbB-2,MMP-2 and MMP-9 proteins,and as compared with control group,the protein expression was decreased significantly in HCMV groups(P<0.05).Primary EVTs could secrete active MMP-2 and MMP-9 in vitro,and the activity of two MMPs was decreased significantly in HCMV groups(P<0.05).The in vitro cell invasion assay showed that the number of primary EVTs permeating Matrigel in HCMV group was decreased(P<0.05).We are led to conclude that HCMV can infect primary EVTs and inhibit their invasion capability,suggesting that the impaired EVT's invasion capability might be related to the abnormal expression of c-erbB-2,MMP-2 and MMP-9 proteins.
This study examined the anti-viral effect of ursolic acid on guinea pig cytomegalovirus (GPCMV) and explored the steps of viral replication targeted by ursolic acid. Cytopathic effect assay and MTT method were employed to determine the 50% cellular cytotoxicity (CC50), 50% effective concentration (EC50) and therapeutic index (TI) with GPCMV. To investigate the specific anti-viral effect of ursolic acid at different temperatures and time points, two other medicines, ganciclovir and Jinyebaidu (JYBD), serving as controls, were studied for comparison. Our results showed that the CC50 of ganciclovir, JYBD and ursolic acid were 333.8, 3015.6, 86.7 μg/mL, respectively; EC50 of ganciclovir, JYBD and ursolic acid was 48.1, 325.5 and 6.8 μg/mL, respectively; TI of ganciclovir, JYBD and ursolic acid was 7, 9, 13, respectively. Similar with ganciclovir, ursolic acid could inhibit the viral synthesis, but did not affect the viral adsorption onto and penetration into cells. We are led to conclude that the anti-cytomegalovirus effect of ursolic acid is significantly stronger than ganciclovir or JYBD, and the cytotoxic effect of ursolic acid lies in its ability to inhibit viral synthesis.
This paper aimed to study the ability of baicalein to block human cytomegalovirus (HCMV) infection in extravillous cytotrophoblasts (EVT) and its effect on the vasoactive intestinal peptide (VIP) expression in HCMV-infected EVT in vitro. A human trophoblast cell line (HPT-8) was chosen in this study. HCMV with 100 TCID50 was added into culture medium to infect HPT-8 cells, and then HCMV pp65 antigen was assayed by immunofluorescence staining. The infection status was determined by virus titration. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect virus DNA load in the infected cells. The expression of VIP mRNA and protein in the infected cells was measured by qRT-PCR, immunocytochemistry and Western blotting. Concentration of VIP secreted in supernatants was determined by ELISA. Red-stained HCMV pp65 antigens were found in infected HPT-8 cells 48 h after infection. HCMV replicated in large quantity in infected HPT-8 cells 4 days after infection, reaching a peak at day 6 post-infection. After treatment with baicalein, virus DNA load in infected HPT-8 cells was decreased (P<0.05), and the levels of VIP mRNA and protein, and the concentration were raised to the normal (P>0.05). Our study suggested that baicalein exerts a positive effect on the VIP expression in HCMV-infected EVT at maternal-fetal interface.
Human trophoblast cells were isolated and cultured in vitro in order to investigate possible pathogenesis of intrauterine infection caused by HCMV.Trophoblast cells were obtained by compound enzymes digestion and discontinuous percoll gradient.Cells and purity were identified by using immunocytochemistry assay with anti-CK7,Vim and β-hCG antibodies.HCMV AD169 strain replication in isolated trophoblast cells and cell apoptosis were detected at different time points post infection(p.i.).The results showed that highly purified trophoblast cells were obtained.Specific virus replication was increased dramatically at the 24th h p.i.,and then increased slowly during 48h and 72h.Apoptosis rate of trophoblast cells infected with HCMV was(34.68±3.14)% at 24th h p.i.,while that in control group was(15.32±2.34)%(P<0.05).It was suggested that highly purified trophoblast cells can be isolated by the simplified cell purification method.HCMV can infect human trophoblast cells,and be quickly replicated,resulting in the accelerated apoptosis of human trophoblast cells during early time.
This study examined the impacts of intrauterine murine cytomegalovirus(MCMV) infection on the long-term learning and memory of offspring.Sexually matured male and female BALB/C mice without MCMV infection were identified by ELISA and then mated.Seventy pregnant mice were randomly divided into the virus group(n=40) and the control group(n=30),in which the pregnant mice were subjected to placenta inoculation of MCMV suspension(1 μL,1×106 PFU) or the same amount of cell culture medium,respectively,at gestational age of 12.5 days.Some pregnant mice [virus group(n=20),control group(n=15)] were sacrificed by cervical dislocation at gestational age of 18.5 days,and the head circumference and brain weight of the mouse fetuses were measured,and the MCMV infection in their brain tissues was detected by PCR.The other pregnant mice [virus group(n=20),control group(n=15)] delivered naturally,and the learning and memory capability of the offspring at 70-day-old was analyzed by Morris water maze test.The results showed that 28.57% mouse fetuses in the virus group developed viral infection in the brain.Their head circumference and brain weight were significantly reduced as compared with those in the control group(P<0.01).The Morris water maze test revealed that the mouse offspring in the control group found the platform with straight-line trajectories after training.In contrast,the counterparts in the virus group intended to enter the central area,but looked for the platform with a circular trajectory.And the infected mice exhibited prolonged swimming distance and swimming latency(P<0.01).It was concluded that:(1) placenta inoculation of MCMV can cause fetal brain infection and intrauterine development retardation;(2) the offspring of MCMV placenta inoculation mice showed a long-term decline in learning and memory capability.
