中华实验和临床病毒学杂志
中華實驗和臨床病毒學雜誌
중화실험화림상병독학잡지
Chinese Journal of Experimental and Clinical Virology
2015年
4期
292-295
,共4页
罗远材%郭路%牛海英%李静
囉遠材%郭路%牛海英%李靜
라원재%곽로%우해영%리정
核因子%乳头状瘤病毒,人%蛋白激酶类%HeLa细胞
覈因子%乳頭狀瘤病毒,人%蛋白激酶類%HeLa細胞
핵인자%유두상류병독,인%단백격매류%HeLa세포
Nuclear factor%Papilloma virus,human%Protein kinases%HeLa cells
目的 比较人乳头状瘤病毒(human papilloma virus,HPV)18型E6蛋白与蛋白激酶R(PKR)对核因子NF-κB p65表达及磷酸化的调控作用.方法 分别构建靶向HPV18E6及PKR基因的短发夹结构RNA干扰序列的重组质粒载体,转染HeLa细胞,以实时荧光定量PCR法检测各组细胞HPV18E6、PKR及NF-κB p65 mRNA的表达,以Western Blot法检测各组细胞HPV18E6、PKR、NF-κB p65蛋白及后两者磷酸化型蛋白的表达,采用SPSS15.0软件包单因素方差分析及Newman-Keuls-q检验比较上述指标在各组间的表达差异.结果 在沉默HPV18E6基因表达的基础上,pSilencer-RNAi-E6转染组PKR mRNA、蛋白及磷酸化型蛋白的相对表达量均高于pSilencer-NC转染组及空白对照组(P＜0.05),而NF-κB p65 mRNA、蛋白及磷酸化型蛋白的相对表达量均低于pSilencer-NC转染组及空白对照组(P＜0.05);在沉默PKR基因表达的基础上,pSilencer-RNAi-PKR转染组NF-κB p65mRNA、蛋白及磷酸化型蛋白的相对表达量与pSilencer-NC转染组及空白对照组比较差异无统计学意义(P＞0.05).结论 HPV18 E6蛋白是HeLa细胞核因子NF-κB p65表达及磷酸化的主要调控者;PKR的表达及磷酸化均受到抑制,对NF-κB p65的调控作用弱.
目的 比較人乳頭狀瘤病毒(human papilloma virus,HPV)18型E6蛋白與蛋白激酶R(PKR)對覈因子NF-κB p65錶達及燐痠化的調控作用.方法 分彆構建靶嚮HPV18E6及PKR基因的短髮夾結構RNA榦擾序列的重組質粒載體,轉染HeLa細胞,以實時熒光定量PCR法檢測各組細胞HPV18E6、PKR及NF-κB p65 mRNA的錶達,以Western Blot法檢測各組細胞HPV18E6、PKR、NF-κB p65蛋白及後兩者燐痠化型蛋白的錶達,採用SPSS15.0軟件包單因素方差分析及Newman-Keuls-q檢驗比較上述指標在各組間的錶達差異.結果 在沉默HPV18E6基因錶達的基礎上,pSilencer-RNAi-E6轉染組PKR mRNA、蛋白及燐痠化型蛋白的相對錶達量均高于pSilencer-NC轉染組及空白對照組(P＜0.05),而NF-κB p65 mRNA、蛋白及燐痠化型蛋白的相對錶達量均低于pSilencer-NC轉染組及空白對照組(P＜0.05);在沉默PKR基因錶達的基礎上,pSilencer-RNAi-PKR轉染組NF-κB p65mRNA、蛋白及燐痠化型蛋白的相對錶達量與pSilencer-NC轉染組及空白對照組比較差異無統計學意義(P＞0.05).結論 HPV18 E6蛋白是HeLa細胞覈因子NF-κB p65錶達及燐痠化的主要調控者;PKR的錶達及燐痠化均受到抑製,對NF-κB p65的調控作用弱.
목적 비교인유두상류병독(human papilloma virus,HPV)18형E6단백여단백격매R(PKR)대핵인자NF-κB p65표체급린산화적조공작용.방법 분별구건파향HPV18E6급PKR기인적단발협결구RNA간우서렬적중조질립재체,전염HeLa세포,이실시형광정량PCR법검측각조세포HPV18E6、PKR급NF-κB p65 mRNA적표체,이Western Blot법검측각조세포HPV18E6、PKR、NF-κB p65단백급후량자린산화형단백적표체,채용SPSS15.0연건포단인소방차분석급Newman-Keuls-q검험비교상술지표재각조간적표체차이.결과 재침묵HPV18E6기인표체적기출상,pSilencer-RNAi-E6전염조PKR mRNA、단백급린산화형단백적상대표체량균고우pSilencer-NC전염조급공백대조조(P＜0.05),이NF-κB p65 mRNA、단백급린산화형단백적상대표체량균저우pSilencer-NC전염조급공백대조조(P＜0.05);재침묵PKR기인표체적기출상,pSilencer-RNAi-PKR전염조NF-κB p65mRNA、단백급린산화형단백적상대표체량여pSilencer-NC전염조급공백대조조비교차이무통계학의의(P＞0.05).결론 HPV18 E6단백시HeLa세포핵인자NF-κB p65표체급린산화적주요조공자;PKR적표체급린산화균수도억제,대NF-κB p65적조공작용약.
Objective To Compare the regulation effects on expression and phosphorylation of nuclear factor NF-κB p65 by human papilloma virus 18 subtype E6 (HPV18E6) protein and protein kinase R (PKR).Methods Constructed two recombinant plasmid vectors which contained shRNA interfering sequence aiming at the targets of HPV18E6 oncogene and PKR gene,and transfected them respectively into HeLa cell,the expression of mRNA of HPV18E6,PKR and NF-κB p65 were determined with real-time quantitative PCR,the expression of protein HPV18E6,PKR,NF-κB p65 and phosphating PKR (P-PKR),phosphating NF-κB p65 (P-NF-κB p65)were determined with Western Blot.The expression differences of these indicators among every group were compared with one-factor analysis of variance and Newman-keuls-q test of statisticalsoftware of SPSS15.0 edition.Results Based on interfering the expression of HPV18E6 oncogene,the expression of PKR mRNA and protein and phosphating protein in pSilencer-RNAi-E6 transfection group were higher than those in pSilencer-NC transfection group and blank control group(P ＜ 0.05),but the expression of NF-κB p65 mRNA and protein and phosphating protein in pSilencer-RNAi-E6 transfection group were lower than those in pSilencer-NC transfection group and blank control group(P ＜ 0.05).Based on interfering the expression of PKR gene,the expression of NF-κB p65 mRNA and protein and phosphating protein in pSilencer-RNAi-PKR transfection group,pSilencer-NC transfection group and blank control group had no significant difference(P ＞0.05).Conclusions HPV18E6 protein was the main regulator of expression and phosphorylation of nuclear factor NF-κB p65 in HeLa cell.The expression and phosphorylation of PKR were both suppressed,so PKR regulated NF-κB p65 weakly in HeLa cell.
