Objective The effect of the functionally unknown gene C6orf120 on autoimmune hepatitis was investigated on C6orf120 knockout rats(C6orf120^(-/-))and THP-1 cells.Method Six–eight-week-old C6orf120^(-/-)and wild-type(W...Objective The effect of the functionally unknown gene C6orf120 on autoimmune hepatitis was investigated on C6orf120 knockout rats(C6orf120^(-/-))and THP-1 cells.Method Six–eight-week-old C6orf120^(-/-)and wild-type(WT)SD rats were injected with Con A(16 mg/kg),and euthanized after 24 h.The sera,livers,and spleens were collected.THP-1 cells and the recombinant protein(rC6ORF120)were used to explore the mechanism in vitro.The frequency of M1 and M2 macrophages was analyzed using flow cytometry.Western blotting and PCR were used to detect macrophage polarization-associated factors.Results C6orf120 knockout attenuated Con A-induced autoimmune hepatitis.Flow cytometry indicated that the proportion of CD68^(+)CD86^(+)M1 macrophages from the liver and spleen in the C6orf120^(-/-)rats decreased.C6orf120 knockout induced downregulation of CD86 protein and the mRNA levels of related inflammatory factors TNF-α,IL-1β,and IL-6 in the liver.C6orf120 knockout did not affect the polarization of THP-1 cells.However,rC6ORF120 promoted the THP-1 cells toward CD68^(+)CD80^(+)M1 macrophages and inhibited the CD68^(+)CD206^(+)M2 phenotype.Conclusion C6orf120 knockout alleviates Con A-induced autoimmune hepatitis by inhibiting macrophage polarization toward M1 macrophages and reducing the expression of related inflammatory factors in C6orf120^(-/-)rats.展开更多
为了给深入研究猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndromevirus,PRRSV)ORF6基因编码的M蛋白的生物学功能提供重要试验材料,本研究首先利用慢病毒包装系统构建了过表达PRRSVORF6基因的重组慢病毒质粒,将该...为了给深入研究猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndromevirus,PRRSV)ORF6基因编码的M蛋白的生物学功能提供重要试验材料,本研究首先利用慢病毒包装系统构建了过表达PRRSVORF6基因的重组慢病毒质粒,将该质粒连同辅助质粒共同转染至HEK293T细胞获得重组慢病毒;之后将重组慢病毒感染MARC-145细胞,利用嘌呤霉素结合有限稀释法进行筛选,连续筛选3轮后建立了稳定表达PRRSVM蛋白的MARC-145ORF6细胞系;并使用CCK-8试验评估过表达PRRSVM蛋白对MARC-145细胞生长的影响。利用RT-PCR、蛋白免疫印迹(Westernblot)和间接免疫荧光(IFA)评估MARC-145ORF6细胞系的传代稳定性并鉴定M蛋白的亚细胞定位,进一步利用RT-qPCR评估过表达M蛋白对MARC-145细胞的干扰素及相关调节基因的影响;此外,还测定了PRRSV在MARC-145ORF6细胞系、MARC-145Flag细胞系和MARC-145细胞中的病毒滴度并绘制多步生长曲线以比较其差异。CCK-8试验结果表明,过表达PRRSVM蛋白对MARC-145细胞活力无显著影响;RT-qPCR、Westernblot和IFA等试验结果表明,MARC-145ORF6细胞系能够表达PRRSV的M蛋白且在传代过程中稳定。此外,稳定表达PRRSVM蛋白显著下调了细胞系的Ⅰ型干扰素及其相关调节基因;多步生长曲线表明,MARC-145ORF6细胞系促进PRRSV增殖,提高其病毒滴度。综上,本研究构建了可以稳定表达PRRSVM蛋白的MARC-145ORF6细胞系,发现其Ⅰ型干扰素水平显著下调且促进PRRSV复制。本研究构建的MARC-145ORF6细胞系将为M蛋白功能的深入研究提供重要生物材料。展开更多
基金supported by the Dengfeng Talent Support Program of Beijing Municipal Administration of Hospitals[Grant No.DFL20221601]the Natural Science Foundation of Beijing[Grant No.7212053]Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine[Grant No.ZYYCXTD-C-202006].
文摘Objective The effect of the functionally unknown gene C6orf120 on autoimmune hepatitis was investigated on C6orf120 knockout rats(C6orf120^(-/-))and THP-1 cells.Method Six–eight-week-old C6orf120^(-/-)and wild-type(WT)SD rats were injected with Con A(16 mg/kg),and euthanized after 24 h.The sera,livers,and spleens were collected.THP-1 cells and the recombinant protein(rC6ORF120)were used to explore the mechanism in vitro.The frequency of M1 and M2 macrophages was analyzed using flow cytometry.Western blotting and PCR were used to detect macrophage polarization-associated factors.Results C6orf120 knockout attenuated Con A-induced autoimmune hepatitis.Flow cytometry indicated that the proportion of CD68^(+)CD86^(+)M1 macrophages from the liver and spleen in the C6orf120^(-/-)rats decreased.C6orf120 knockout induced downregulation of CD86 protein and the mRNA levels of related inflammatory factors TNF-α,IL-1β,and IL-6 in the liver.C6orf120 knockout did not affect the polarization of THP-1 cells.However,rC6ORF120 promoted the THP-1 cells toward CD68^(+)CD80^(+)M1 macrophages and inhibited the CD68^(+)CD206^(+)M2 phenotype.Conclusion C6orf120 knockout alleviates Con A-induced autoimmune hepatitis by inhibiting macrophage polarization toward M1 macrophages and reducing the expression of related inflammatory factors in C6orf120^(-/-)rats.
文摘为了给深入研究猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndromevirus,PRRSV)ORF6基因编码的M蛋白的生物学功能提供重要试验材料,本研究首先利用慢病毒包装系统构建了过表达PRRSVORF6基因的重组慢病毒质粒,将该质粒连同辅助质粒共同转染至HEK293T细胞获得重组慢病毒;之后将重组慢病毒感染MARC-145细胞,利用嘌呤霉素结合有限稀释法进行筛选,连续筛选3轮后建立了稳定表达PRRSVM蛋白的MARC-145ORF6细胞系;并使用CCK-8试验评估过表达PRRSVM蛋白对MARC-145细胞生长的影响。利用RT-PCR、蛋白免疫印迹(Westernblot)和间接免疫荧光(IFA)评估MARC-145ORF6细胞系的传代稳定性并鉴定M蛋白的亚细胞定位,进一步利用RT-qPCR评估过表达M蛋白对MARC-145细胞的干扰素及相关调节基因的影响;此外,还测定了PRRSV在MARC-145ORF6细胞系、MARC-145Flag细胞系和MARC-145细胞中的病毒滴度并绘制多步生长曲线以比较其差异。CCK-8试验结果表明,过表达PRRSVM蛋白对MARC-145细胞活力无显著影响;RT-qPCR、Westernblot和IFA等试验结果表明,MARC-145ORF6细胞系能够表达PRRSV的M蛋白且在传代过程中稳定。此外,稳定表达PRRSVM蛋白显著下调了细胞系的Ⅰ型干扰素及其相关调节基因;多步生长曲线表明,MARC-145ORF6细胞系促进PRRSV增殖,提高其病毒滴度。综上,本研究构建了可以稳定表达PRRSVM蛋白的MARC-145ORF6细胞系,发现其Ⅰ型干扰素水平显著下调且促进PRRSV复制。本研究构建的MARC-145ORF6细胞系将为M蛋白功能的深入研究提供重要生物材料。
