目的建立用大鼠胶质瘤细胞系(Rat glial cell line C6)替代大鼠原代胚细胞(Primary rat embryo cells,RE)来培养大鼠细小病毒(Kilham Rat Virus,KRV)的方法。方法将500 TCID50 KRV培养物接种到75T-C6细胞培养瓶中(细胞接种量为2×10...目的建立用大鼠胶质瘤细胞系(Rat glial cell line C6)替代大鼠原代胚细胞(Primary rat embryo cells,RE)来培养大鼠细小病毒(Kilham Rat Virus,KRV)的方法。方法将500 TCID50 KRV培养物接种到75T-C6细胞培养瓶中(细胞接种量为2×105/mL),培养过夜,待细胞病变CPE达++～+++时,分别用免疫荧光(FITC)鉴定所培养病毒的特异抗原,用血球凝集试验(HA)测定培养物上清的效价,用DNA测序鉴定所培养的病毒,最后用96孔板培养法测定KRV的TCID50。结果 KRV在接种到C6细胞的第4～5天,细胞发生明显的病变,CPE可达++++,FITC鉴定呈KRV抗原阳性,病毒的培养上清中HA效价为1∶5 120,测序结果表明,该病毒序列与NCBI中KRV序列同源性达98%,确定为KRV。收获的KRV的TCID50为104.6/0.1 mL。结论通过对C6细胞系培养KRV方法的标准化和对所培养病毒的一系列鉴定表明,用大鼠胶质瘤细胞系可以替代大鼠原代胚细胞系进行大鼠细小病毒的培养。展开更多
The inhibitory effect of parvovirus H-1 on the colonyforming ability in vitro of QGY-7703, a cultured human hepatoma cell line, and on the formation and growth of its tumors in nude mice was studied. With higher multi...The inhibitory effect of parvovirus H-1 on the colonyforming ability in vitro of QGY-7703, a cultured human hepatoma cell line, and on the formation and growth of its tumors in nude mice was studied. With higher multiplicity of infection (MOI) of H-1 given, survival of the QGY-7703 cells was found to be decreased. H-1 DNA amplification level at 30 h postinfection(p.i.) was detected to be 7.4 times higher than that at 2 h by dispersed cells assay, while the cells were delayed to enter into S phase.Plaques were formed in the indicator cells (new-born human kidney cell line, NBK) by progeny H-1 virus particles released from the infected QGY-7703 cells by infectious cell center assay. The formation of tumors in nude mice by QGY-7703 cells which were injected s c at 2 h postinfection was observed to be prevented in 2 groups with given MOI 25 and 50. The tumor growth of MOI 10 group occurred at a lower exponential rate than that of control,after a 20 d latent period. It was evident that parvovirus H-1 exhibited a direct inhibitory effect on the formation and growth of human hepatoma cells in vivo as well as in vitro.展开更多
A group of SARS-like coronaviruses(SL-CoV)have been identified in horseshoe bats.Despite SL-CoVs and SARS-CoV share identical genome structure and high-level sequence similarity,SL-CoV does not bind to the same cellul...A group of SARS-like coronaviruses(SL-CoV)have been identified in horseshoe bats.Despite SL-CoVs and SARS-CoV share identical genome structure and high-level sequence similarity,SL-CoV does not bind to the same cellular receptor as for SARS-CoV and the N-terminus of the S proteins only share 64%amino acid identity,suggesting there are fundamental differences between these two groups of coronaviruses.To gain insight into the basis of this difference,we established a recombinant adenovirus system expressing the S protein from SL-CoV(rAd-Rp3-S)to investigate its immune characterization.Our results showed that immunized mice generated strong humoral immune responses against the SL-CoV S protein.Moreover,a strong cellular immune response demonstrated by elevated IFN-γand IL-6 levels was also observed in these mice.However,the induced antibody from these mice had weaker cross-reaction with the SARS-CoV S protein,and did not neutralize HIV pseudotyped with SARS-CoV S protein.These results demonstrated that the immunogenicity of the SL-CoV S protein is distinct from that of SARS-CoV,which may cause the immunological differences between human SARS-CoV and bat SL-CoV.Furthermore,the recombinant virus could serve as a potential vaccine candidate against bat SL-CoV infection.展开更多
The aim of this study is to investigate the effects of allitridin on the expression of transcription factor T-bet/GATA-3 in mice infected by murine cytomegalovirus. BALB/c mice model system of murine cytomegalovirus (...The aim of this study is to investigate the effects of allitridin on the expression of transcription factor T-bet/GATA-3 in mice infected by murine cytomegalovirus. BALB/c mice model system of murine cytomegalovirus (MCMV) infection was established. In which 20 model mice were allocated randomly into allitridin treated group ( n =10) and infected control group ( n =10). Allitridin (25 mg·kg -1 ·d -1 ) was used in treated group at the 24 h by intraperitoneal route ( once/d ×14 d), and the same volume of saline solution was injected control mice. Normal control mice ( n =10), were only given with the same volume of 0.89% sodium chloride, without infection with MCMV. The expression levels of transcription factor T-bet/GATA-3 mRNA were measured by RT-PCR, and the expression levels of T helper 1(Th1) cytokine IFN-γ and Th2 cytokine IL-10 in supernatant of spleen cell culture were measured by ELISA. Experimental results showed MCMV infection could markedly down-modulate the expression of IFN-γ and T-bet, and significantly up-modulate the expression of IL-10 and GATA-3 mRNA. Allitridin could induce increased expression of transcription factor T-bet mRNA and Th1 cytokine IFN-γ significantly ( P <0.01), and decreased expression of transcription factor GATA-3 mRNA and Th2 cytokine IL-10 markedly ( P <0.01). It is concluded that MCMV infection leads to disequilibrium of Th1/Th2 cytokine expression: the level of Th1 cytokine IFN-γ decreases significantly and Th2 cytokine IL-10 overexpresses markedly. Allitridin can up-regulate the expression of T-bet and IFN-γ, and inhibit the expression of GATA-3 mRNA and IL-10 in MCMV infected mice, indicating a Th1 dominant state which should enhance the specific cellular immune reactions against CMV and be helpful for clearance of the cytomegalovirus in host.展开更多
Functional human hepatocytes xenografted into the liver of mice can be used as a model system to study pharmacokinetics,infection of hepatitis viruses,and the efficacy of hepatitis vaccines.Significant levels of liver...Functional human hepatocytes xenografted into the liver of mice can be used as a model system to study pharmacokinetics,infection of hepatitis viruses,and the efficacy of hepatitis vaccines.Significant levels of liver xeno-repopulation have been reported in Fah-/-Rag2-/-Il2rg-/-mice.However,the high mortality and low breeding rate of this model may hinder its application.A new model,termed Fah-/-Nod/Scid mice,which combines the advantages of liver repopulation in Fah-/-mice with the ease of xenotransplantation in Nod/Scid mice was obtained by gradual cross-breeding.Fah-/-Nod/Scid mice were easily maintained in breeding colonies and in adult animal care facilities.FK506 treatment combined with gradual withdrawal of NTBC before cell transplantation ensured that Fah-/-Nod/Scid mice were susceptible to liver xeno-repopulation by human hepatocytes;the proportion of engrafted human hepatocytes reached 33.6%.The function of the expanded human hepatocytes within the chimeric liver was confirmed by weight curve analysis,the expression of characteristic proteins,and the biochemical analysis of liver function.These results show that Fah-/-Nod/Scid mice are an ideal humanized liver mouse model with many useful applications.展开更多
文摘目的建立用大鼠胶质瘤细胞系(Rat glial cell line C6)替代大鼠原代胚细胞(Primary rat embryo cells,RE)来培养大鼠细小病毒(Kilham Rat Virus,KRV)的方法。方法将500 TCID50 KRV培养物接种到75T-C6细胞培养瓶中(细胞接种量为2×105/mL),培养过夜,待细胞病变CPE达++～+++时,分别用免疫荧光(FITC)鉴定所培养病毒的特异抗原,用血球凝集试验(HA)测定培养物上清的效价,用DNA测序鉴定所培养的病毒,最后用96孔板培养法测定KRV的TCID50。结果 KRV在接种到C6细胞的第4～5天,细胞发生明显的病变,CPE可达++++,FITC鉴定呈KRV抗原阳性,病毒的培养上清中HA效价为1∶5 120,测序结果表明,该病毒序列与NCBI中KRV序列同源性达98%,确定为KRV。收获的KRV的TCID50为104.6/0.1 mL。结论通过对C6细胞系培养KRV方法的标准化和对所培养病毒的一系列鉴定表明,用大鼠胶质瘤细胞系可以替代大鼠原代胚细胞系进行大鼠细小病毒的培养。
文摘The inhibitory effect of parvovirus H-1 on the colonyforming ability in vitro of QGY-7703, a cultured human hepatoma cell line, and on the formation and growth of its tumors in nude mice was studied. With higher multiplicity of infection (MOI) of H-1 given, survival of the QGY-7703 cells was found to be decreased. H-1 DNA amplification level at 30 h postinfection(p.i.) was detected to be 7.4 times higher than that at 2 h by dispersed cells assay, while the cells were delayed to enter into S phase.Plaques were formed in the indicator cells (new-born human kidney cell line, NBK) by progeny H-1 virus particles released from the infected QGY-7703 cells by infectious cell center assay. The formation of tumors in nude mice by QGY-7703 cells which were injected s c at 2 h postinfection was observed to be prevented in 2 groups with given MOI 25 and 50. The tumor growth of MOI 10 group occurred at a lower exponential rate than that of control,after a 20 d latent period. It was evident that parvovirus H-1 exhibited a direct inhibitory effect on the formation and growth of human hepatoma cells in vivo as well as in vitro.
基金supported by the State Key Program for Basic Research Grant(2005CB523004)from the Chinese Ministry of Science and Technologythe Knowledge Innovation Program Key Project administered by the Chinese Academy of Sciences(KSCX1-YW-R-07)
文摘A group of SARS-like coronaviruses(SL-CoV)have been identified in horseshoe bats.Despite SL-CoVs and SARS-CoV share identical genome structure and high-level sequence similarity,SL-CoV does not bind to the same cellular receptor as for SARS-CoV and the N-terminus of the S proteins only share 64%amino acid identity,suggesting there are fundamental differences between these two groups of coronaviruses.To gain insight into the basis of this difference,we established a recombinant adenovirus system expressing the S protein from SL-CoV(rAd-Rp3-S)to investigate its immune characterization.Our results showed that immunized mice generated strong humoral immune responses against the SL-CoV S protein.Moreover,a strong cellular immune response demonstrated by elevated IFN-γand IL-6 levels was also observed in these mice.However,the induced antibody from these mice had weaker cross-reaction with the SARS-CoV S protein,and did not neutralize HIV pseudotyped with SARS-CoV S protein.These results demonstrated that the immunogenicity of the SL-CoV S protein is distinct from that of SARS-CoV,which may cause the immunological differences between human SARS-CoV and bat SL-CoV.Furthermore,the recombinant virus could serve as a potential vaccine candidate against bat SL-CoV infection.
文摘The aim of this study is to investigate the effects of allitridin on the expression of transcription factor T-bet/GATA-3 in mice infected by murine cytomegalovirus. BALB/c mice model system of murine cytomegalovirus (MCMV) infection was established. In which 20 model mice were allocated randomly into allitridin treated group ( n =10) and infected control group ( n =10). Allitridin (25 mg·kg -1 ·d -1 ) was used in treated group at the 24 h by intraperitoneal route ( once/d ×14 d), and the same volume of saline solution was injected control mice. Normal control mice ( n =10), were only given with the same volume of 0.89% sodium chloride, without infection with MCMV. The expression levels of transcription factor T-bet/GATA-3 mRNA were measured by RT-PCR, and the expression levels of T helper 1(Th1) cytokine IFN-γ and Th2 cytokine IL-10 in supernatant of spleen cell culture were measured by ELISA. Experimental results showed MCMV infection could markedly down-modulate the expression of IFN-γ and T-bet, and significantly up-modulate the expression of IL-10 and GATA-3 mRNA. Allitridin could induce increased expression of transcription factor T-bet mRNA and Th1 cytokine IFN-γ significantly ( P <0.01), and decreased expression of transcription factor GATA-3 mRNA and Th2 cytokine IL-10 markedly ( P <0.01). It is concluded that MCMV infection leads to disequilibrium of Th1/Th2 cytokine expression: the level of Th1 cytokine IFN-γ decreases significantly and Th2 cytokine IL-10 overexpresses markedly. Allitridin can up-regulate the expression of T-bet and IFN-γ, and inhibit the expression of GATA-3 mRNA and IL-10 in MCMV infected mice, indicating a Th1 dominant state which should enhance the specific cellular immune reactions against CMV and be helpful for clearance of the cytomegalovirus in host.
基金supported by the National High Technology Research and Development Program of China(Grant No. 2006AA02Z474)the National Natural Science Foundation of China(Grant No. 30801115)+1 种基金the China Postdoctoral Science Foundation(Grant No. 20070410743)the National Basic Research Program of China(Grant No. 2010CB945600)
文摘Functional human hepatocytes xenografted into the liver of mice can be used as a model system to study pharmacokinetics,infection of hepatitis viruses,and the efficacy of hepatitis vaccines.Significant levels of liver xeno-repopulation have been reported in Fah-/-Rag2-/-Il2rg-/-mice.However,the high mortality and low breeding rate of this model may hinder its application.A new model,termed Fah-/-Nod/Scid mice,which combines the advantages of liver repopulation in Fah-/-mice with the ease of xenotransplantation in Nod/Scid mice was obtained by gradual cross-breeding.Fah-/-Nod/Scid mice were easily maintained in breeding colonies and in adult animal care facilities.FK506 treatment combined with gradual withdrawal of NTBC before cell transplantation ensured that Fah-/-Nod/Scid mice were susceptible to liver xeno-repopulation by human hepatocytes;the proportion of engrafted human hepatocytes reached 33.6%.The function of the expanded human hepatocytes within the chimeric liver was confirmed by weight curve analysis,the expression of characteristic proteins,and the biochemical analysis of liver function.These results show that Fah-/-Nod/Scid mice are an ideal humanized liver mouse model with many useful applications.
