核糖体结合蛋白1干扰慢病毒载体的构建及其对人肝癌细胞的干扰效应
|
摘要
目的构建核糖体结合蛋白1(RPN1)的干扰慢病毒载体,建立下调RPN1表达的肝癌细胞系。方法运用分子克隆针对RPN1基因构建干扰慢病毒载体,通过与病毒包装辅助质粒共感染293T细胞包装病毒,高速离心浓缩病毒,免疫荧光方式测定病毒滴度,感染Huh7.5.1肝癌细胞系,实时荧光定量PCR(RTqPCR)测定病毒干扰效果。结果成功构建并包装RPN1RNA干扰慢病毒,病毒滴度为2×10~9 TU/mL,感染Huh7.5.1细胞后阳性率大于90.0%。相对于阴性对照,RPN1RNA干扰慢病毒组RPN1基因表达敲减效率达到92.4%(P<0.05)。结论成功构建并包装了RPN1干扰慢病毒,建立下调RPN1表达的人肝癌细胞株。
Objective To construct a interference lentivirus targeting ribophorin 1(RPN1),and establish a hepatocellular carcinoma cell line that down-regulates the expression of RPN1.Methods A interference lentivirus vector targeting the RPN1 gene was constructed by molecular cloning.The interference lentivirus was packed from 293 T cells by coinfection with virus packaging auxiliary plasmids.After high speed centrifugation,the virus was concentrated and its titer was detected by immunofluorescence.The hepatoma cell line Huh7.5.1 was infected with the interference lentivirus and the interference effect of the virus was determined by RT-qPCR.Results The RPN1 RNA interference lentivirus was successfully constructed and packaged.The titer of virus was 2×10~9 TU/mL,and the positive rate after infection of Huh7.5.1 cells was more than 90.0%.Compared with negative control,the knockdown rate of RPN1 gene expression in RPN1 RNA interference lentivirus group was 92.4%(P<0.05).Conclusion RPN1 interfering lentivirus was successfully constructed and packaged,and a human hepatocellular carcinoma cell line with down-regulation of RPN1 expression was established.
Objective To construct a interference lentivirus targeting ribophorin 1(RPN1),and establish a hepatocellular carcinoma cell line that down-regulates the expression of RPN1.Methods A interference lentivirus vector targeting the RPN1 gene was constructed by molecular cloning.The interference lentivirus was packed from 293 T cells by coinfection with virus packaging auxiliary plasmids.After high speed centrifugation,the virus was concentrated and its titer was detected by immunofluorescence.The hepatoma cell line Huh7.5.1 was infected with the interference lentivirus and the interference effect of the virus was determined by RT-qPCR.Results The RPN1 RNA interference lentivirus was successfully constructed and packaged.The titer of virus was 2×10~9 TU/mL,and the positive rate after infection of Huh7.5.1 cells was more than 90.0%.Compared with negative control,the knockdown rate of RPN1 gene expression in RPN1 RNA interference lentivirus group was 92.4%(P<0.05).Conclusion RPN1 interfering lentivirus was successfully constructed and packaged,and a human hepatocellular carcinoma cell line with down-regulation of RPN1 expression was established.
引文
[1]TORRE L A,BRAY F,SIEGEL R L,et al.Global cancer statistics,2012[J].CA Cancer J Clin,2015,65(2):87-108.
[2]CHEN W Q,ZHENG R S,BAADE P D,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-132.
[3]聂笠.慢性乙型和丙型病毒性肝炎合并原发性肝癌的相关因素研究[D].北京:中国疾病预防控制中心,2017.
[4]WILSON C M,ROEBUCK Q,HIGH S.RibophorinⅠregulates substrate delivery to the oligosaccharyltransferase core[J].Proc Natl Acad Sci U S A,2008,105(28):9534-9539.
[5]TAKEDA K,QIN S Y,MATSUMOTO N,et al.Association of malectin with ribophorin I is crucial for attenuation of misfolded glycoprotein secretion[J].Biochem Biophys Res Commun,2014,454(3):436-440.
[6]MOLTHATHONG S,BUAKLIN A,SENAPIN S A,et al.Up-regulation of ribophorin l after yellow head virus(YHV)challenge in black tiger shrimp penaeus monodon[J].Fish Shellfish Immunol,2008,25(1/2):40-46.
[7]MARCEAU C D,PUSCHNIK A S,MAJZOUB K,et al.Genetic dissection of Flaviviridae host factors through genomescale CRISPR screens[J].Nature,2016,535(7610):159.
[8]HOLMES D R,KEREIAKES D J,GARG S,et al.Stent thrombosis[J].J Am Coll Cardiol,2010,56(17):1357.
[9]董孟权,杨丽英.核糖体结合蛋白在甲状腺癌中的表达及其与临床病理特征的关系[J].蚌埠医学院学报,2016,41(2):215-216.
[10]董孟权.浅论核糖体结合蛋白在不同甲状腺微小癌患者中的表达关系[J].医药卫生(文摘版),2015,7(12):00012.
[11]卢明芹,杨敏.丙型肝炎病毒相关性肝癌的研究进展[J].现代医药卫生,2017,33(20):3080-3082.
[12]秦健,李文静,冯勇,等.乙型肝炎,丙型肝炎病毒相关性肝癌基因差异表达的生物信息学分析[J].武汉大学学报(医学版),2017,38(4):604-609.
[13]曾洁,黄天壬,邓伟.乙型肝炎病毒X蛋白与内质网应激关系的研究进展[J].病毒学报,2018,34(2):272-276.
[14]潘玲,张翠薇,马程功,等.内质网应激诱导自噬的分子机制[J].现代生物医学进展,2017,17(13):2590-2593.
[15]钟强,陈小伍,朱达坚.内质网应激与肿瘤关系的研究进展[J].广东医学,2015,36(12):1937-1939.
[16]王晋蜀,王伟佳,王婷,等.ICAT干扰慢病毒表达载体构建及稳转HL60细胞株的建立[J].国际检验医学杂志,2016,37(7):875-877.
[17]宁慧宇,刘才,邹华文.基因功能缺失技术及研究现状[J].湖北农业科学,2017,56(8):1405-1412.
[18]蒲小勇,周香雪,肖恒军,等.短发夹RNA靶向沉默大鼠阴茎海绵体平滑肌细胞胰岛素样生长因子结合蛋白-3的研究[J].中华实验外科杂志,2017,34(8):228.
[19]RAO D D,VORHIES J S,SENZER N,er al.siRNAvs.shRNA:similarities and differences[J].Adv Drug Deliv Rev,2009,61(9):746-759.
[20]隋玉飞,刘乃政,司磊.慢病毒载体介导survivin基因siRNA对裸鼠移植人肺腺癌的体内抗癌机制研究[J].国际检验医学杂志,2016,37(5):583-585.
[21]文扬,朱亚琴.PERK干扰慢病毒载体的构建及其在人牙髓细胞中的表达[J].上海口腔医学,2017,26(4):363-367.
[22]房超,尹晶,于秋爽,等.慢病毒介导的靶向干扰MEX3A基因膀胱癌稳定细胞株的建立[J].中国医科大学学报,2017,46(12):1057-1061.
[2]CHEN W Q,ZHENG R S,BAADE P D,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-132.
[3]聂笠.慢性乙型和丙型病毒性肝炎合并原发性肝癌的相关因素研究[D].北京:中国疾病预防控制中心,2017.
[4]WILSON C M,ROEBUCK Q,HIGH S.RibophorinⅠregulates substrate delivery to the oligosaccharyltransferase core[J].Proc Natl Acad Sci U S A,2008,105(28):9534-9539.
[5]TAKEDA K,QIN S Y,MATSUMOTO N,et al.Association of malectin with ribophorin I is crucial for attenuation of misfolded glycoprotein secretion[J].Biochem Biophys Res Commun,2014,454(3):436-440.
[6]MOLTHATHONG S,BUAKLIN A,SENAPIN S A,et al.Up-regulation of ribophorin l after yellow head virus(YHV)challenge in black tiger shrimp penaeus monodon[J].Fish Shellfish Immunol,2008,25(1/2):40-46.
[7]MARCEAU C D,PUSCHNIK A S,MAJZOUB K,et al.Genetic dissection of Flaviviridae host factors through genomescale CRISPR screens[J].Nature,2016,535(7610):159.
[8]HOLMES D R,KEREIAKES D J,GARG S,et al.Stent thrombosis[J].J Am Coll Cardiol,2010,56(17):1357.
[9]董孟权,杨丽英.核糖体结合蛋白在甲状腺癌中的表达及其与临床病理特征的关系[J].蚌埠医学院学报,2016,41(2):215-216.
[10]董孟权.浅论核糖体结合蛋白在不同甲状腺微小癌患者中的表达关系[J].医药卫生(文摘版),2015,7(12):00012.
[11]卢明芹,杨敏.丙型肝炎病毒相关性肝癌的研究进展[J].现代医药卫生,2017,33(20):3080-3082.
[12]秦健,李文静,冯勇,等.乙型肝炎,丙型肝炎病毒相关性肝癌基因差异表达的生物信息学分析[J].武汉大学学报(医学版),2017,38(4):604-609.
[13]曾洁,黄天壬,邓伟.乙型肝炎病毒X蛋白与内质网应激关系的研究进展[J].病毒学报,2018,34(2):272-276.
[14]潘玲,张翠薇,马程功,等.内质网应激诱导自噬的分子机制[J].现代生物医学进展,2017,17(13):2590-2593.
[15]钟强,陈小伍,朱达坚.内质网应激与肿瘤关系的研究进展[J].广东医学,2015,36(12):1937-1939.
[16]王晋蜀,王伟佳,王婷,等.ICAT干扰慢病毒表达载体构建及稳转HL60细胞株的建立[J].国际检验医学杂志,2016,37(7):875-877.
[17]宁慧宇,刘才,邹华文.基因功能缺失技术及研究现状[J].湖北农业科学,2017,56(8):1405-1412.
[18]蒲小勇,周香雪,肖恒军,等.短发夹RNA靶向沉默大鼠阴茎海绵体平滑肌细胞胰岛素样生长因子结合蛋白-3的研究[J].中华实验外科杂志,2017,34(8):228.
[19]RAO D D,VORHIES J S,SENZER N,er al.siRNAvs.shRNA:similarities and differences[J].Adv Drug Deliv Rev,2009,61(9):746-759.
[20]隋玉飞,刘乃政,司磊.慢病毒载体介导survivin基因siRNA对裸鼠移植人肺腺癌的体内抗癌机制研究[J].国际检验医学杂志,2016,37(5):583-585.
[21]文扬,朱亚琴.PERK干扰慢病毒载体的构建及其在人牙髓细胞中的表达[J].上海口腔医学,2017,26(4):363-367.
[22]房超,尹晶,于秋爽,等.慢病毒介导的靶向干扰MEX3A基因膀胱癌稳定细胞株的建立[J].中国医科大学学报,2017,46(12):1057-1061.