AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of...AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.展开更多
AIM:To investigate the expression of the hepatitis B virus(HBV)1.3-fold genome plasmid(pHBV1.3)in an immortalized mouse hepatic cell line induced by SV40T-antigen(SV40T)expression.METHODS:Mouse hepatic cells were isol...AIM:To investigate the expression of the hepatitis B virus(HBV)1.3-fold genome plasmid(pHBV1.3)in an immortalized mouse hepatic cell line induced by SV40T-antigen(SV40T)expression.METHODS:Mouse hepatic cells were isolated from mouse liver tissue fragments from 3-5 d old Kunming mice by the direct collagenase digestion method and cultured in vitro.The pRSV-T plasmid was transfected into mouse hepatic cells to establish an SV40LT-immortalized mouse hepatic cell line.The SV40LT-immortalized mouse hepatic cells were identified and transfected with the pHBV1.3 plasmid.The levels of hepatitis B surface antigen(HBsAg)and hepatitis B e antigen(HBeAg)in the supernatant were determined by an electrochemiluminescence immunoassay at 24,48,72 and 96 h after transfection.The expressions of HBsAg and hepatitis B c antigen(HBcAg)in the cells were investigated by indirect immunofluorescence analysis.The presence of HBV DNA replication intermediates in the transfected cells and viral particles in the supernatant of the transfected cell cultures was monitored using the Southern hybridization assay and transmission electronic microscopy,respectively.RESULTS:The pRSV-T plasmid was used to immortalize mouse hepatocytes and an SV40LT-immortalized mouse hepatic cell line was successfully established.SV40LT-immortalized mouse hepatic cells have the same morphology and growth characteristics as primary mouse hepatic cells can be subcultured and produce albumin and cytokeratin-18 in vitro.Immortalized mouse hepatic cells did not show the characteristics of tumor cells,as alpha-fetoprotein levels were comparable(0.58±0.37 vs 0.61±0.31,P=0.37).SV40LTimmortalized mouse hepatic cells were then transfected with the pHBV1.3 plasmid,and it was found that the HBV genome replicated in SV40LT-immortalized mouse hepatic cells.The levels of HBsAg and HBeAg continuously increased in the supernatant after the transfection of pHBV1.3,and began to decrease 72 h after transfection.The expressions of HBsAg and HBcAg were observed in the pHBV1.3-transfected cells.HBV DNA replication intermediates were also observed at72 h after transfection,including relaxed circular DNA,double-stranded DNA and single-stranded DNA.Furthermore,a few 42 nm Dane particles,as well as many22 nm subviral particles with a spherical or filamentous shape,were detected in the supernatant.CONCLUSION:SV40T expression can immortalize mouse hepatic cells,and the pHBV1.3-transfected SV40T-immortalized mouse hepatic cell line can be a new in vitro cell model.展开更多
目的建立稳定的SV40感染滴度测定方法,制备高滴度SV40,用于生物制品病毒清除/灭活工艺的验证。方法通过分析不同细胞感染SV40后出现病变的时间、病变程度及产毒量,确定SV40敏感细胞株。分析维持液、细胞培养时间、病毒吸附时间等对病毒...目的建立稳定的SV40感染滴度测定方法,制备高滴度SV40,用于生物制品病毒清除/灭活工艺的验证。方法通过分析不同细胞感染SV40后出现病变的时间、病变程度及产毒量,确定SV40敏感细胞株。分析维持液、细胞培养时间、病毒吸附时间等对病毒滴定测定的影响,建立SV40滴度测定的方法。并分析病毒感染后不同时间及细胞不同部位SV40滴度的差异,制备大量的高滴度SV40。结果与Vero、Vero76及VeroE6细胞相比,CV-1细胞对SV40高度敏感,细胞病变出现时间最早,病变最明显,产毒量最高。SV40滴度与病毒接种前细胞的培养时间、细胞接种量和维持液无明显相关,但吸附时间对病毒滴度有一定的影响。病毒的最佳吸附时间为120 min。接种病毒后48 h收集细胞沉淀,所获得的SV40滴度最高,平均为8.81 lg CCID50/ml。结论已建立了稳定的SV40滴度测定方法,并制备了高滴度SV40,为病毒清除/灭活工艺验证研究奠定了基础。展开更多
目的建立稳定分化大鼠骨骺软骨细胞株,为细胞替代治疗和基因治疗小儿生长发育迟缓提供稳定的细胞来源。方法利用脂质体介导的基因转染技术将含有猿肾病毒40大T抗原(simian virus 40 large T antigen gene,SV40LTag)基因的质粒pEGFP-IRES...目的建立稳定分化大鼠骨骺软骨细胞株,为细胞替代治疗和基因治疗小儿生长发育迟缓提供稳定的细胞来源。方法利用脂质体介导的基因转染技术将含有猿肾病毒40大T抗原(simian virus 40 large T antigen gene,SV40LTag)基因的质粒pEGFP-IRES2-SV40LTag转染原代培养的新生大鼠骨骺软骨细胞,G418筛选,抗性克隆扩大培养传代。应用II型胶原、X型胶原和SV40LTag抗体进行细胞鉴定,体外检测其分化能力,观察细胞的形态及其生长状况,绘制细胞生长曲线。用RT-PCR、Southern blot和免疫细胞化学法鉴定SV40LTag在转染细胞中的表达。结果转染后获得了阳性细胞克隆,免疫细胞化学证实为具有较强增殖能力和多分化潜能的骨骺软骨细胞。经Southern印迹杂交证实,SV40LTag已稳定转染入骨骺软骨细胞,表达mRNA及其蛋白。结论SV40LTag导入可诱导骨骺软骨细胞稳定分化,为细胞替代治疗和基因治疗小儿生长发育迟缓等疾病提供稳定的细胞来源。展开更多
基金Supported by National Natural Science Foundation of China(No.81273212,81100651)Project of Science and Technology of Shandong Province(No.2014GSF118044)
文摘AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.
基金Supported by Jinan Science and Technology Bureau,Shandong Province,China,No.200705095-4
文摘AIM:To investigate the expression of the hepatitis B virus(HBV)1.3-fold genome plasmid(pHBV1.3)in an immortalized mouse hepatic cell line induced by SV40T-antigen(SV40T)expression.METHODS:Mouse hepatic cells were isolated from mouse liver tissue fragments from 3-5 d old Kunming mice by the direct collagenase digestion method and cultured in vitro.The pRSV-T plasmid was transfected into mouse hepatic cells to establish an SV40LT-immortalized mouse hepatic cell line.The SV40LT-immortalized mouse hepatic cells were identified and transfected with the pHBV1.3 plasmid.The levels of hepatitis B surface antigen(HBsAg)and hepatitis B e antigen(HBeAg)in the supernatant were determined by an electrochemiluminescence immunoassay at 24,48,72 and 96 h after transfection.The expressions of HBsAg and hepatitis B c antigen(HBcAg)in the cells were investigated by indirect immunofluorescence analysis.The presence of HBV DNA replication intermediates in the transfected cells and viral particles in the supernatant of the transfected cell cultures was monitored using the Southern hybridization assay and transmission electronic microscopy,respectively.RESULTS:The pRSV-T plasmid was used to immortalize mouse hepatocytes and an SV40LT-immortalized mouse hepatic cell line was successfully established.SV40LT-immortalized mouse hepatic cells have the same morphology and growth characteristics as primary mouse hepatic cells can be subcultured and produce albumin and cytokeratin-18 in vitro.Immortalized mouse hepatic cells did not show the characteristics of tumor cells,as alpha-fetoprotein levels were comparable(0.58±0.37 vs 0.61±0.31,P=0.37).SV40LTimmortalized mouse hepatic cells were then transfected with the pHBV1.3 plasmid,and it was found that the HBV genome replicated in SV40LT-immortalized mouse hepatic cells.The levels of HBsAg and HBeAg continuously increased in the supernatant after the transfection of pHBV1.3,and began to decrease 72 h after transfection.The expressions of HBsAg and HBcAg were observed in the pHBV1.3-transfected cells.HBV DNA replication intermediates were also observed at72 h after transfection,including relaxed circular DNA,double-stranded DNA and single-stranded DNA.Furthermore,a few 42 nm Dane particles,as well as many22 nm subviral particles with a spherical or filamentous shape,were detected in the supernatant.CONCLUSION:SV40T expression can immortalize mouse hepatic cells,and the pHBV1.3-transfected SV40T-immortalized mouse hepatic cell line can be a new in vitro cell model.
文摘目的建立稳定的SV40感染滴度测定方法,制备高滴度SV40,用于生物制品病毒清除/灭活工艺的验证。方法通过分析不同细胞感染SV40后出现病变的时间、病变程度及产毒量,确定SV40敏感细胞株。分析维持液、细胞培养时间、病毒吸附时间等对病毒滴定测定的影响,建立SV40滴度测定的方法。并分析病毒感染后不同时间及细胞不同部位SV40滴度的差异,制备大量的高滴度SV40。结果与Vero、Vero76及VeroE6细胞相比,CV-1细胞对SV40高度敏感,细胞病变出现时间最早,病变最明显,产毒量最高。SV40滴度与病毒接种前细胞的培养时间、细胞接种量和维持液无明显相关,但吸附时间对病毒滴度有一定的影响。病毒的最佳吸附时间为120 min。接种病毒后48 h收集细胞沉淀,所获得的SV40滴度最高,平均为8.81 lg CCID50/ml。结论已建立了稳定的SV40滴度测定方法,并制备了高滴度SV40,为病毒清除/灭活工艺验证研究奠定了基础。
文摘目的建立稳定分化大鼠骨骺软骨细胞株,为细胞替代治疗和基因治疗小儿生长发育迟缓提供稳定的细胞来源。方法利用脂质体介导的基因转染技术将含有猿肾病毒40大T抗原(simian virus 40 large T antigen gene,SV40LTag)基因的质粒pEGFP-IRES2-SV40LTag转染原代培养的新生大鼠骨骺软骨细胞,G418筛选,抗性克隆扩大培养传代。应用II型胶原、X型胶原和SV40LTag抗体进行细胞鉴定,体外检测其分化能力,观察细胞的形态及其生长状况,绘制细胞生长曲线。用RT-PCR、Southern blot和免疫细胞化学法鉴定SV40LTag在转染细胞中的表达。结果转染后获得了阳性细胞克隆,免疫细胞化学证实为具有较强增殖能力和多分化潜能的骨骺软骨细胞。经Southern印迹杂交证实,SV40LTag已稳定转染入骨骺软骨细胞,表达mRNA及其蛋白。结论SV40LTag导入可诱导骨骺软骨细胞稳定分化,为细胞替代治疗和基因治疗小儿生长发育迟缓等疾病提供稳定的细胞来源。
