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 beta-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 48 h and 72 h. 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.

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 complex phosphoesterasum digestion and gradient centrifugation from villous tissue aseptically taken from healthy pregnant women. Cytokeratin7 (CK7), vimentin (Vim) and c-erbB-2 were immunocytochemically 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.

OBJECTIVETo study the in vitro anti-human cytomegalovirus effect and the cytotoxicity of Forsythia suspensa and its main active ingredient quercetin.

METHODThe 0% toxic dose (TD0), minimum effective concentration (MEC) and therapeutic index (TI) of anti-human cytomegalovirus activity by F. suspensa and quercetin were detected with the cytopathic assay and MTT method. Ganciclovir was used as the control drug for comparison.

RESULTThe TD0 of ganciclovir, F. suspensa and quercetin were 10, 30, 30 mg L(-1), the MEC were 10, 30, 0.3 mg x L(-1), TI were 1, 1 and 100, respectively.

CONCLUSIONThe anti-human cytomegalovirus effect of quercetin is much higher than ganciclovir and F. suspensa, and the cytotoxicity is equivalent to F. suspensa but lower than ganciclovir. Therefore, quercetin has the potential advantages of anti-human cytomegalovirus effect.

Antiviral Agents ; pharmacology ; toxicity ; Cell Line ; Cytomegalovirus ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; toxicity ; Forsythia ; chemistry ; Humans ; Quercetin ; pharmacology ; toxicity

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.
ADAM Proteins ; blood ; metabolism ; ADAMTS13 Protein ; Adult ; Blood Coagulation ; physiology ; Case-Control Studies ; Female ; Humans ; Pre-Eclampsia ; blood ; enzymology ; physiopathology ; Pregnancy ; Pregnancy Trimester, Third ; Young Adult ; von Willebrand Factor ; metabolism

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 48 h and 72 h.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=l5)] 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.

Objective:To understand the current research status of master of professional degree students in clinical medicine in provincial teaching hospitals with moderate scientific research level under the " dual-track" training system.Methods:Sojump online survey was conducted to investigate the cognition, current situation and self-evaluation of all clinical master degree students in three grades in a provincial teaching hospital, and the research status of professional degree and scientific degree students was compared and analyzed respectively.Results:The proportion of scientific degree students participating in scientific research projects was significantly higher than that of professional degree students. The proportion of professional degree students participating in scientific research projects was still not high even in the third year of graduate students. However, there was no difference between scientific degree and professional degree students in the publication of scientific research papers. The scientific degree of scientific research knowledge is significantly higher than that of professional degree students. Although scientific degree students receive more scientific research guidance from their supervisors, professional degree students communicate more with their supervisors, and the results show that professional degree students are significantly more satisfied with their supervisors, graduate policies and scientific research policies than scientific degree students. In addition, the results of graduate students found that the degree of research stress of both scientific and professional degrees exceeded 50%.Conclusions:Scientific degree is better than professional degree in research status because of professional characteristics and more research guidance from their supervisors. However, through the reform of professional degree training mode based on scientific research project management in recent years, professional degree students have been able to communicate more with their supervisors, and their satisfaction with their supervisors and colleges has significantly increased. In addition, sufficient attention should be paid to high scientific research pressure of medical graduate students.

The effect of human cytomegalovirus (HCMV) on invasive capability of early pregnant extravillous cytotrophoblasts (EVTs) was investigated in vitro.Primary EVTs were obtained by complex phosphoesterasum digestion and gradient centrifugation from villous tissue aseptically taken from healthy pregnant women.Cytokeratin7 (CK7),vimentin (Vim) and c-erbB-2 were immunocytochemically 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-2and MMP-9 proteins.