[Objective] The study aimed at cloning PKR gene from Ctenopharyngodon idellus induced by PolyI：C in vitro,so as to provide foundation for study on the anti-virus genes of C.idellus.[Method] By referring to the PKR ge...[Objective] The study aimed at cloning PKR gene from Ctenopharyngodon idellus induced by PolyI：C in vitro,so as to provide foundation for study on the anti-virus genes of C.idellus.[Method] By referring to the PKR gene sequences of zebra fish（AJ852023.1） and Carassius auratus（AY293929.1） in Genbank,three pairs of degenerate primers were designed with Primer Premier 5.0 software;in vitro C.idellus kidney cells（CIK） were treated with 100 μg/ml of Poly I：C for 12,36 and 48 h,and then total RNA of the cells treated was extracted for amplifying the PKR gene by RT-PCR.[Result] The PKR gene was amplified from the cells treated with Poly I：C for 36 and 48 h,but not from the cells treated for 12 h;in addition,the expression level increased with the processing time.Part of the amplified sequence of C.idellus shared the homology of 100% and 81.48% with the sequences of carp and zebra fish separately.[Conclusion] Part of the PKR gene sequence was cloned successfully from C.idellus.Moreover,we have proved that PolyI：C induction is effective for PKR protein expression,which will provide reference for treating viral diseases of C.idellus.展开更多
探讨polyI:C是否影响小鼠蜕膜基质细胞存活及其细胞因子的产生,为进一步阐明双链RNA或病毒诱导早孕期母体流产的潜在机制奠定基础。分离培养小鼠蜕膜基质细胞,polyI:C处理细胞24 h后,采用噻唑盐比色法(MTT)检测细胞存活率,Annexin V FIT...探讨polyI:C是否影响小鼠蜕膜基质细胞存活及其细胞因子的产生,为进一步阐明双链RNA或病毒诱导早孕期母体流产的潜在机制奠定基础。分离培养小鼠蜕膜基质细胞,polyI:C处理细胞24 h后,采用噻唑盐比色法(MTT)检测细胞存活率,Annexin V FITC流式细胞术(FCM)分析polyI:C诱导细胞的死亡方式,以及应用实时定量PCR分析polyI:C处理后IFN-β的表达情况。MTT检测结果表明2 mg/L polyI:C处理细胞后,细胞存活率显著降低(P<0.01),Annexin V FITC流式检测说明polyI:C诱导蜕膜基质细胞的细胞死亡方式以坏死和晚期凋亡为主,并且polyI:C可显著促进IFN-β的表达。提示双链RNA能显著促进小鼠蜕膜基质细胞的死亡和细胞因子IFN-β的表达。展开更多
基金Supported by National Natural Science Foundation of Zhejiang Province (Y3110432 )Huzhou Teachers College Science ResearchFoundation (2010YZ48)~~
文摘[Objective] The study aimed at cloning PKR gene from Ctenopharyngodon idellus induced by PolyI：C in vitro,so as to provide foundation for study on the anti-virus genes of C.idellus.[Method] By referring to the PKR gene sequences of zebra fish（AJ852023.1） and Carassius auratus（AY293929.1） in Genbank,three pairs of degenerate primers were designed with Primer Premier 5.0 software;in vitro C.idellus kidney cells（CIK） were treated with 100 μg/ml of Poly I：C for 12,36 and 48 h,and then total RNA of the cells treated was extracted for amplifying the PKR gene by RT-PCR.[Result] The PKR gene was amplified from the cells treated with Poly I：C for 36 and 48 h,but not from the cells treated for 12 h;in addition,the expression level increased with the processing time.Part of the amplified sequence of C.idellus shared the homology of 100% and 81.48% with the sequences of carp and zebra fish separately.[Conclusion] Part of the PKR gene sequence was cloned successfully from C.idellus.Moreover,we have proved that PolyI：C induction is effective for PKR protein expression,which will provide reference for treating viral diseases of C.idellus.
文摘探讨polyI:C是否影响小鼠蜕膜基质细胞存活及其细胞因子的产生,为进一步阐明双链RNA或病毒诱导早孕期母体流产的潜在机制奠定基础。分离培养小鼠蜕膜基质细胞,polyI:C处理细胞24 h后,采用噻唑盐比色法(MTT)检测细胞存活率,Annexin V FITC流式细胞术(FCM)分析polyI:C诱导细胞的死亡方式,以及应用实时定量PCR分析polyI:C处理后IFN-β的表达情况。MTT检测结果表明2 mg/L polyI:C处理细胞后,细胞存活率显著降低(P<0.01),Annexin V FITC流式检测说明polyI:C诱导蜕膜基质细胞的细胞死亡方式以坏死和晚期凋亡为主,并且polyI:C可显著促进IFN-β的表达。提示双链RNA能显著促进小鼠蜕膜基质细胞的死亡和细胞因子IFN-β的表达。
