AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcin...AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination. RESULTS: The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes. CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.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 The Major Scientific and Technological Project of Hubei Province, No. 2007ABD005
文摘AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination. RESULTS: The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes. CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.
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。
