Increasing evidence has revealed that maternal cytomegalovirus （CMV） infection may be associated with neurodevelopmental disorders in offspring. Potential relevance between the placental inflammation and CMV-related...Increasing evidence has revealed that maternal cytomegalovirus （CMV） infection may be associated with neurodevelopmental disorders in offspring. Potential relevance between the placental inflammation and CMV-related autism has been reported by clinical observation. Meanwhile, abnormal expression of Toll-like receptor 2 （TLR2） and TLR4 in placenta of patients with chorioamnionitis was observed in multiple studies. IL-6 and IL- 10 are two important maternal inflammatory mediators involved in neurodevelopmental disorders. To investigate whether murine CMV （MCMV） infection causes alterations in placental IL-6/10 and TLR2/4 levels, we analyzed the dynamic changes in gene expression of TLR2/4 and IL-6/10 in placentas following acute MCMV infection. Mouse model of acute MCMV infection during pregnancy was created, and pre-pregnant MCMV infected, lipopolysaccharide （LPS）-treated and uninfected mice were used as controls. At E13.5, E 14.5 and E 18.5, placentas and fetal brains were harvested and mRNA expression levels of placental TLR2/4 and IL-6/10 were analyzed. The results showed that after acute MCMV infection, the expression levels of placental TLR2/4 and IL-6 were elevated at E13.5, accompanied by obvious placental inflammation and reduction of placenta and fetal brain weights. However, LPS 50 ktg/kg could decrease the IL-6 expression at E13.5 and E14.5. This suggests that acute MCMV infection during pregnancy could up-regulate the gene expression of TLR2/4 in placental trophoblasts and activate them to produce more pro- inflammatory cytokine IL-6. High dose of LPS stimulation （50 gg/kg） during pregnancy can lead to down-regulation of IL-6 levels in the late stage. Imbalance of IL-6 expression in placenta might be associated with the neurodevelopmental disorders in progeny.展开更多
In order to explore the effects of testicular infection of murine cytomegalovirus （MCMV） on mature sperm viability at different periods following MCMV inoculation in mice, 91 BALB/c mice without MCMV infection were ...In order to explore the effects of testicular infection of murine cytomegalovirus （MCMV） on mature sperm viability at different periods following MCMV inoculation in mice, 91 BALB/c mice without MCMV infection were randomly divided into two groups： an experimental group （n= 56） and a control group （n=35）. The mice in the experimental group were treated by inoculating MCMV intratesticularly, while those in the controlled group were directly inoculated with DMEM without MCMV. The mice in both groups were sacrificed separately on the day 1,1.5, 2, 4, 6, 9 and 14 post-inoculation （D1, 1.5, 2, 4, 6, 9 and 14 PI）. The MCMV M83 mRNA gene was detected in the testis by in situ hybridization （ISH） with MCMV late-mRNA probe labeled with digoxin. Sperm viability of mature sperm in the epididymis cauda was measured. The results demonstrated the positive signal of ISH of MCMV was found mainly in the cytoplasm of the testicular interstitial cells and spermatogenic cells in the experimental group. Compared with that in the controlled group, the sperm viability in the experimental group was decreased significantly on D1 PI and D1.5 PI （P〈 0.05）. No statistically significant difference in the sperm viability was found after D2 PI between two groups （P〉0.05）. This suggested that sperm viability in mice might be descended significantly shortly after MCMV infection and might return to normal with time, indicating that MCMV acute infection might temporarily degrade sperm quality and influence procreation transiently.展开更多
In order to establish a model system of the murine hepatocyte infection by murine cytomegalovirus (MCMV), the primary cultured murine hepatocytes were obtained in a modified low serum medium system by a non perfusio...In order to establish a model system of the murine hepatocyte infection by murine cytomegalovirus (MCMV), the primary cultured murine hepatocytes were obtained in a modified low serum medium system by a non perfusion method, and then infected by Smith strain MCMV. Infected hepatocytes showed characteristic cytopathic effect (CPE) at 30 h after infection, in which a large number of viral particles was found and ultrastructures were destroyed (as revealed by disappearance of bile canalicula and organelles) under the electron microscope and MCMV immediate early genes were detected by in situ hybridization. Meanwhile, infected cells produced albumin significantly less than corresponding uninfected controls. On the contrary, uninfected controls simultaneously cultured under the same conditions showed normal function and ultratructure (glycogen rosettes, bile canalicula, wheel like mitochondria and well developed rough and smooth endoplasmic reticula). These results demonstrated that a model system of primary cultured murine hepatocytes infected by MCMV was successfully set up.展开更多
Objective: A BALB/c mice model of cytomegalovirus-induced myocarditis was established. Methods: Twenty-five inbred female BALB/c mice free of murine cytomegalovirus(MCMV) infection (5 weeks old, 16-18 g),were infected...Objective: A BALB/c mice model of cytomegalovirus-induced myocarditis was established. Methods: Twenty-five inbred female BALB/c mice free of murine cytomegalovirus(MCMV) infection (5 weeks old, 16-18 g),were infected with 1×10~4 PFU MCMV by the intraperitoneal (i.p.) administration. All experimental mice were sacrificed on day 3, 5, 7, 10,and 14 after i. p. administration. The hearts were removed under aseptic conditions, and were transected along the midline. Aliquots of hearts were handled with Bouin's fixative for histological examination. Residual hearts were immediately frozen in liquid nitrogen and stored at -80℃ until MCMV titre was determined by a plaque assay. Seurm cTnI level was assayed by ELISA. Results: MCMV in the heart was at extremely low level on day 3 after i. p. administration, reached to the peak on day 7-10, and then ran down. A mixed cellular infiltrate composed of polymorphonuclear neutrophils and mononuclear lymphocytes was observed on day 3, reaching to the peak on day 7-10 after MCMV infection, and was maintained for at least 3-4 months later. Seurm cTnI levels were elevated on day 3 after i.p. administration, reaching to the peak it day 7-10. Conclusion: The BALB/c mice model for cytomegalovirus-induced myocarditis was successfully established, that might make it possible to screen antiviral drugs for treating viral myocarditis and to investigate and evaluate the pathogenesis and prognosis of this disease.展开更多
Absent in melanoma 2(AIM2)inflammasome is a crucial link bridging the innate host defense and the subsequent adaptive immunity when activated by exogenous double stranded DNA(dsDNA).Through establishing models of diss...Absent in melanoma 2(AIM2)inflammasome is a crucial link bridging the innate host defense and the subsequent adaptive immunity when activated by exogenous double stranded DNA(dsDNA).Through establishing models of disseminated murine cytomegalovirus(MCMV)infection in BALB/c and C57BL/6 mice,we evaluated dynamic expression of AIM2 inflammasome components and its relationship with pathological damage and viral replication,trying tofigure out whether AIM2 inflammasome is related to the chronic mechanism of MCMV.BALB/c and C57BL/6 mice were sacrificed on day 0,1,3,7,14 and 28 post infection.Expression levels of AIM2,pro-caspase-1,caspase-1 p20,pro-IL1β and mature IL1β in primary peritoneal macrophages(PMs)and spleens were detected by Western blotting.Contents of IL18 in the serum were detected by ELISA.Pathological examinations of livers were performed,and mRNA levels of MCMV glycoprotein B(gB)in salivary glands also assessed.Results showed that expression levels of AIM2 in PMs and spleens of C57BL/6 mice increased on day 3,even continued to day 28;caspase-1 p20 and mature IL1β increased on day 7,14 and 28;the persistently high expression of IL1β in the serum started on day 1,showing a double peak curve.As for BALB/c mice,expression of AIM2 in PMs increased on day 1 and day 7,while contents of AIM2 in spleens increased on day 1 and day 3;caspase-1 p20 and mature ILip merely increased 7 days fter infection.Thereafter,expression levels of AIM2,caspase-1 p20,mature IL1β and IL18 were limited;the duration of AIM2 inflammasome activation in BALB/c mice was much shorter than that in C57BL/6 mice.The severer pathological damage and more viral replications in BALB/c mice further proved the deficient antiviral immunity to MCMV.In conclusion,the activation of AIM2 inflammasome in BALB/c mice was short-lived,which is quite possibly related to the chronicity of MCMV infection.展开更多
文摘Increasing evidence has revealed that maternal cytomegalovirus （CMV） infection may be associated with neurodevelopmental disorders in offspring. Potential relevance between the placental inflammation and CMV-related autism has been reported by clinical observation. Meanwhile, abnormal expression of Toll-like receptor 2 （TLR2） and TLR4 in placenta of patients with chorioamnionitis was observed in multiple studies. IL-6 and IL- 10 are two important maternal inflammatory mediators involved in neurodevelopmental disorders. To investigate whether murine CMV （MCMV） infection causes alterations in placental IL-6/10 and TLR2/4 levels, we analyzed the dynamic changes in gene expression of TLR2/4 and IL-6/10 in placentas following acute MCMV infection. Mouse model of acute MCMV infection during pregnancy was created, and pre-pregnant MCMV infected, lipopolysaccharide （LPS）-treated and uninfected mice were used as controls. At E13.5, E 14.5 and E 18.5, placentas and fetal brains were harvested and mRNA expression levels of placental TLR2/4 and IL-6/10 were analyzed. The results showed that after acute MCMV infection, the expression levels of placental TLR2/4 and IL-6 were elevated at E13.5, accompanied by obvious placental inflammation and reduction of placenta and fetal brain weights. However, LPS 50 ktg/kg could decrease the IL-6 expression at E13.5 and E14.5. This suggests that acute MCMV infection during pregnancy could up-regulate the gene expression of TLR2/4 in placental trophoblasts and activate them to produce more pro- inflammatory cytokine IL-6. High dose of LPS stimulation （50 gg/kg） during pregnancy can lead to down-regulation of IL-6 levels in the late stage. Imbalance of IL-6 expression in placenta might be associated with the neurodevelopmental disorders in progeny.
文摘In order to explore the effects of testicular infection of murine cytomegalovirus （MCMV） on mature sperm viability at different periods following MCMV inoculation in mice, 91 BALB/c mice without MCMV infection were randomly divided into two groups： an experimental group （n= 56） and a control group （n=35）. The mice in the experimental group were treated by inoculating MCMV intratesticularly, while those in the controlled group were directly inoculated with DMEM without MCMV. The mice in both groups were sacrificed separately on the day 1,1.5, 2, 4, 6, 9 and 14 post-inoculation （D1, 1.5, 2, 4, 6, 9 and 14 PI）. The MCMV M83 mRNA gene was detected in the testis by in situ hybridization （ISH） with MCMV late-mRNA probe labeled with digoxin. Sperm viability of mature sperm in the epididymis cauda was measured. The results demonstrated the positive signal of ISH of MCMV was found mainly in the cytoplasm of the testicular interstitial cells and spermatogenic cells in the experimental group. Compared with that in the controlled group, the sperm viability in the experimental group was decreased significantly on D1 PI and D1.5 PI （P〈 0.05）. No statistically significant difference in the sperm viability was found after D2 PI between two groups （P〉0.05）. This suggested that sperm viability in mice might be descended significantly shortly after MCMV infection and might return to normal with time, indicating that MCMV acute infection might temporarily degrade sperm quality and influence procreation transiently.
文摘In order to establish a model system of the murine hepatocyte infection by murine cytomegalovirus (MCMV), the primary cultured murine hepatocytes were obtained in a modified low serum medium system by a non perfusion method, and then infected by Smith strain MCMV. Infected hepatocytes showed characteristic cytopathic effect (CPE) at 30 h after infection, in which a large number of viral particles was found and ultrastructures were destroyed (as revealed by disappearance of bile canalicula and organelles) under the electron microscope and MCMV immediate early genes were detected by in situ hybridization. Meanwhile, infected cells produced albumin significantly less than corresponding uninfected controls. On the contrary, uninfected controls simultaneously cultured under the same conditions showed normal function and ultratructure (glycogen rosettes, bile canalicula, wheel like mitochondria and well developed rough and smooth endoplasmic reticula). These results demonstrated that a model system of primary cultured murine hepatocytes infected by MCMV was successfully set up.
文摘Objective: A BALB/c mice model of cytomegalovirus-induced myocarditis was established. Methods: Twenty-five inbred female BALB/c mice free of murine cytomegalovirus(MCMV) infection (5 weeks old, 16-18 g),were infected with 1×10~4 PFU MCMV by the intraperitoneal (i.p.) administration. All experimental mice were sacrificed on day 3, 5, 7, 10,and 14 after i. p. administration. The hearts were removed under aseptic conditions, and were transected along the midline. Aliquots of hearts were handled with Bouin's fixative for histological examination. Residual hearts were immediately frozen in liquid nitrogen and stored at -80℃ until MCMV titre was determined by a plaque assay. Seurm cTnI level was assayed by ELISA. Results: MCMV in the heart was at extremely low level on day 3 after i. p. administration, reached to the peak on day 7-10, and then ran down. A mixed cellular infiltrate composed of polymorphonuclear neutrophils and mononuclear lymphocytes was observed on day 3, reaching to the peak on day 7-10 after MCMV infection, and was maintained for at least 3-4 months later. Seurm cTnI levels were elevated on day 3 after i.p. administration, reaching to the peak it day 7-10. Conclusion: The BALB/c mice model for cytomegalovirus-induced myocarditis was successfully established, that might make it possible to screen antiviral drugs for treating viral myocarditis and to investigate and evaluate the pathogenesis and prognosis of this disease.
基金the National Natural Science Foundation of China(No.81271807).
文摘Absent in melanoma 2(AIM2)inflammasome is a crucial link bridging the innate host defense and the subsequent adaptive immunity when activated by exogenous double stranded DNA(dsDNA).Through establishing models of disseminated murine cytomegalovirus(MCMV)infection in BALB/c and C57BL/6 mice,we evaluated dynamic expression of AIM2 inflammasome components and its relationship with pathological damage and viral replication,trying tofigure out whether AIM2 inflammasome is related to the chronic mechanism of MCMV.BALB/c and C57BL/6 mice were sacrificed on day 0,1,3,7,14 and 28 post infection.Expression levels of AIM2,pro-caspase-1,caspase-1 p20,pro-IL1β and mature IL1β in primary peritoneal macrophages(PMs)and spleens were detected by Western blotting.Contents of IL18 in the serum were detected by ELISA.Pathological examinations of livers were performed,and mRNA levels of MCMV glycoprotein B(gB)in salivary glands also assessed.Results showed that expression levels of AIM2 in PMs and spleens of C57BL/6 mice increased on day 3,even continued to day 28;caspase-1 p20 and mature IL1β increased on day 7,14 and 28;the persistently high expression of IL1β in the serum started on day 1,showing a double peak curve.As for BALB/c mice,expression of AIM2 in PMs increased on day 1 and day 7,while contents of AIM2 in spleens increased on day 1 and day 3;caspase-1 p20 and mature ILip merely increased 7 days fter infection.Thereafter,expression levels of AIM2,caspase-1 p20,mature IL1β and IL18 were limited;the duration of AIM2 inflammasome activation in BALB/c mice was much shorter than that in C57BL/6 mice.The severer pathological damage and more viral replications in BALB/c mice further proved the deficient antiviral immunity to MCMV.In conclusion,the activation of AIM2 inflammasome in BALB/c mice was short-lived,which is quite possibly related to the chronicity of MCMV infection.
