中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2013年
24期
1906-1910
,共5页
王轩%周益%袁伟杰%尚明花%包谨芳%朱楠
王軒%週益%袁偉傑%尚明花%包謹芳%硃楠
왕헌%주익%원위걸%상명화%포근방%주남
受体,Notch1%肝炎病毒,乙型%肾小管,近端%上皮细胞%免疫分子
受體,Notch1%肝炎病毒,乙型%腎小管,近耑%上皮細胞%免疫分子
수체,Notch1%간염병독,을형%신소관,근단%상피세포%면역분자
Receptor,Notch1%Hepatitis B virus%Kidney tubules,proximal%Epithelial cells%Immune molecules
目的 探讨乙型肝炎病毒X基因(HBx)转染人近端肾小管上皮细胞系HK-2细胞后对其表达Notch1信号的影响,并观察Notch1信号在HBx介导的肾小管上皮细胞免疫活性中的作用.方法 用分子克隆的方法构建pcDNA3.1/myc-HBx质粒及pcDNA3.1/myc-Notch1质粒,转染HK-2细胞,并应用短发卡RNA(shRNA)沉默Notch1基因表达.将细胞分为7组:①正常培养组；②转染HBx空载质粒组；③转染HBx质粒组；④转染HBx质粒+Notch1空载质粒组；⑤转染HBx质粒+Notch1质粒组；⑥转染HBx质粒+Notch1 shRNA空载质粒组；⑦转染HBx质粒+Notch1 shRNA沉默质粒组.实时定量PCR和蛋白印迹法检测细胞中Notch1受体的变化,流式细胞术检测细胞表面人主要组织相容性复合物(MHC)-Ⅱ、CD40表达的变化,酶联免疫吸附试验(ELISA)检测细胞培养上清中白细胞介素(IL)4、干扰素(IFN)-γ水平.结果 HK-2细胞经转染HBx基因后,可高表达HBx,Notch1shRNA在HK-2细胞中的转染效率也高达70％,基因沉默有效；转染HBx基因组细胞Notch1水平高于正常培养组及转染HBx空载质粒组相比,且Notch1过表达组细胞表面MHC-Ⅱ、CD40表达均明显高于Notch1空载质粒组(9.69％±0.52％比4.90％±0.32％,21.56％ ±0.71％比15.74％±0.20％,均P ＜0.05),其上清液中IL-4水平亦高于Notch1空载质粒组(50.2±0.6比28.1±1.2,P＜0.05),而IFN-γ水平较低(11.9±1.7比18.8±0.8,P＜0.05).Notch1基因沉默后上述指标结果呈相反变化.结论 HBx基因转染HK-2细胞后可引起Notch1表达上调；Notch1又可促进细胞表面免疫分子的表达,并调控细胞因子分泌,最终导致细胞损伤及免疫微环境的紊乱.
目的 探討乙型肝炎病毒X基因(HBx)轉染人近耑腎小管上皮細胞繫HK-2細胞後對其錶達Notch1信號的影響,併觀察Notch1信號在HBx介導的腎小管上皮細胞免疫活性中的作用.方法 用分子剋隆的方法構建pcDNA3.1/myc-HBx質粒及pcDNA3.1/myc-Notch1質粒,轉染HK-2細胞,併應用短髮卡RNA(shRNA)沉默Notch1基因錶達.將細胞分為7組:①正常培養組；②轉染HBx空載質粒組；③轉染HBx質粒組；④轉染HBx質粒+Notch1空載質粒組；⑤轉染HBx質粒+Notch1質粒組；⑥轉染HBx質粒+Notch1 shRNA空載質粒組；⑦轉染HBx質粒+Notch1 shRNA沉默質粒組.實時定量PCR和蛋白印跡法檢測細胞中Notch1受體的變化,流式細胞術檢測細胞錶麵人主要組織相容性複閤物(MHC)-Ⅱ、CD40錶達的變化,酶聯免疫吸附試驗(ELISA)檢測細胞培養上清中白細胞介素(IL)4、榦擾素(IFN)-γ水平.結果 HK-2細胞經轉染HBx基因後,可高錶達HBx,Notch1shRNA在HK-2細胞中的轉染效率也高達70％,基因沉默有效；轉染HBx基因組細胞Notch1水平高于正常培養組及轉染HBx空載質粒組相比,且Notch1過錶達組細胞錶麵MHC-Ⅱ、CD40錶達均明顯高于Notch1空載質粒組(9.69％±0.52％比4.90％±0.32％,21.56％ ±0.71％比15.74％±0.20％,均P ＜0.05),其上清液中IL-4水平亦高于Notch1空載質粒組(50.2±0.6比28.1±1.2,P＜0.05),而IFN-γ水平較低(11.9±1.7比18.8±0.8,P＜0.05).Notch1基因沉默後上述指標結果呈相反變化.結論 HBx基因轉染HK-2細胞後可引起Notch1錶達上調；Notch1又可促進細胞錶麵免疫分子的錶達,併調控細胞因子分泌,最終導緻細胞損傷及免疫微環境的紊亂.
목적 탐토을형간염병독X기인(HBx)전염인근단신소관상피세포계HK-2세포후대기표체Notch1신호적영향,병관찰Notch1신호재HBx개도적신소관상피세포면역활성중적작용.방법 용분자극륭적방법구건pcDNA3.1/myc-HBx질립급pcDNA3.1/myc-Notch1질립,전염HK-2세포,병응용단발잡RNA(shRNA)침묵Notch1기인표체.장세포분위7조:①정상배양조；②전염HBx공재질립조；③전염HBx질립조；④전염HBx질립+Notch1공재질립조；⑤전염HBx질립+Notch1질립조；⑥전염HBx질립+Notch1 shRNA공재질립조；⑦전염HBx질립+Notch1 shRNA침묵질립조.실시정량PCR화단백인적법검측세포중Notch1수체적변화,류식세포술검측세포표면인주요조직상용성복합물(MHC)-Ⅱ、CD40표체적변화,매련면역흡부시험(ELISA)검측세포배양상청중백세포개소(IL)4、간우소(IFN)-γ수평.결과 HK-2세포경전염HBx기인후,가고표체HBx,Notch1shRNA재HK-2세포중적전염효솔야고체70％,기인침묵유효；전염HBx기인조세포Notch1수평고우정상배양조급전염HBx공재질립조상비,차Notch1과표체조세포표면MHC-Ⅱ、CD40표체균명현고우Notch1공재질립조(9.69％±0.52％비4.90％±0.32％,21.56％ ±0.71％비15.74％±0.20％,균P ＜0.05),기상청액중IL-4수평역고우Notch1공재질립조(50.2±0.6비28.1±1.2,P＜0.05),이IFN-γ수평교저(11.9±1.7비18.8±0.8,P＜0.05).Notch1기인침묵후상술지표결과정상반변화.결론 HBx기인전염HK-2세포후가인기Notch1표체상조；Notch1우가촉진세포표면면역분자적표체,병조공세포인자분비,최종도치세포손상급면역미배경적문란.
Objective To explore the expression of Notch1 receptor in renal tubular epithelial cells transfected with hepatitis B virus X (HBx) gene and its role in immunologic activity.Methods The eukaryotic vector pcDNA3.1/myc-HBx containing HBx gene or vector pcDNA3.1/myc-Notch1 containing Notch1 gene was transiently transfected into HK-2 cells.And the shRNA technique was used to silence Notch1.HK-2 cells were divided into 7 groups,including normal culture,pcDNA3.1/myc,HBx,HBx + pcDNA3.1/myc,HBx + Notch1,HBx + shRNA and HBx + Notch1 shRNA.Real-time PCR and Western blotting were used to detect the expression of Notch1.The expressions of MHC-Ⅱ and CD40 were examined by flow cytometry.And the supernatant contents of IL-4 and IFN-γ were measured by enzyme-linked immunosorbent assay (ELISA).Results The results of real-time PCR and Western blotting verified that HBx and Notch1 were successfully expressed in HK-2 cells after transfection.The transfection efficiency of shRNA was 70％.Compared with normal culture and pcDNA3.1/myc groups,the expression of Notch1 increased.The expressions of MHC-Ⅱ and CD40 also significantly increased in the HBx + Notch1 group (9.69％ ±0.52％ vs4.90％ ±0.32％,21.56％ ±0.71％ vs 15.74％ ±0.20％,bothP＜0.05).The supernatant level of IFN-γwas lower in HBx + Notch1 group (11.9 ± 1.7 vs 18.8 ± 0.8,P ＜ 0.05) while the level of IL-4 was higher than control groups (50.2 ±0.6 vs 28.1 ± 1.2,P ＜0.05).And the HBx + Notch1 shRNA group had the opposite results.Conclusions An over-expression of HBx gene may upregulate the expression of Notch1.And Notch1 promotes the expression of immune molecules of renal tubular epithelial cell and regulates the secretion of cytokines so as to cause injury of cells and dysfunction of immunomicroenviroment.
