经HBx转染后的肾小管上皮细胞对T细胞激活、分化的影响
경HBx전염후적신소관상피세포대T세포격활、분화적영향
Effect of renal tubular epithelial cells with hepatitis B virus x gene on mediating T-cell activation and differentiation
  • 万方数据
    中华肾脏病杂志 중화신장병잡지

    2013年 7期 503-508 ,共6页

    王轩%周益%袁伟杰%朱楠%尚明花%赵亚鹏%王玲%谷立杰

    왕헌%주익%원위걸%주남%상명화%조아붕%왕령%곡립걸

    肝炎病毒,乙型%基因%细胞增殖%T淋巴细胞,辅助诱导%肾小管上皮细胞%共刺激分子 肝炎病毒,乙型%基因%細胞增殖%T淋巴細胞,輔助誘導%腎小管上皮細胞%共刺激分子
    간염병독,을형%기인%세포증식%T림파세포,보조유도%신소관상피세포%공자격분자
    Hepatitis B virus%Genes%Cell proliferation%T-lymphocytes,helper-inducer%Renal tubular epithelial cells%Costimulatory molecules
    目的 探讨乙型肝炎病毒x基因(hepatitis B virus x,HBx)转染人近端肾小管上皮细胞系(HK-2)后其对T细胞激活、分化的影响.方法 分4组:实验组(即转染HBx质粒的HK-2细胞+CD4+T细胞)、阴性对照组(即转染HBx空载质粒的HK-2细胞+CD4+T细胞)、单独培养组(即CD4+T细胞)和空白对照组(未处理HK-2细胞+CD4+T细胞).体外培养HK-2,用分子克隆的方法构建pcDNA3.1-myc-HBx质粒,采用脂质体转染法瞬时转染HK-2细胞,实时荧光定量PCR及Western印迹法验证HBx在HK-2细胞中的表达.免疫磁珠法分选健康志愿者外周血CD4+T细胞,分别与HK-2细胞共培养,流式细胞术检测HK-2细胞共刺激分子CD40的表达、CD4+T细胞CD40配体(CD40L)表达及细胞周期情况,ELISA检测各组细胞培养上清中Th1型细胞因子IFN-γ及Th2型细胞因子IL-4水平.结果 HK-2细胞经转染pcDNA3.1-myc-HBx质粒后,可高表达HBx.转染HBx基因后HK-2细胞CD40表达显著上调(P＜0.01)；与对照组相比,实验组CD4+T细胞表面CD40L表达也显著增加(P＜0.01),S期与G2/M期细胞数之和增多(P＜0.01),培养上清中IFN-γ水平升高(P＜0.05),IL-4水平降低(P＜0.05).结论 肾小管上皮细胞经HBx转染后,其共刺激分子表达上调,并促进CD4+T细胞增殖,诱导CD4+T细胞向Th1方向分化.由此推测,乙肝相关性肾炎肾组织免疫损伤所导致病变的持续进展也可能与此因素的参与相关.
    목적 탐토을형간염병독x기인(hepatitis B virus x,HBx)전염인근단신소관상피세포계(HK-2)후기대T세포격활、분화적영향.방법 분4조:실험조(즉전염HBx질립적HK-2세포+CD4+T세포)、음성대조조(즉전염HBx공재질립적HK-2세포+CD4+T세포)、단독배양조(즉CD4+T세포)화공백대조조(미처리HK-2세포+CD4+T세포).체외배양HK-2,용분자극륭적방법구건pcDNA3.1-myc-HBx질립,채용지질체전염법순시전염HK-2세포,실시형광정량PCR급Western인적법험증HBx재HK-2세포중적표체.면역자주법분선건강지원자외주혈CD4+T세포,분별여HK-2세포공배양,류식세포술검측HK-2세포공자격분자CD40적표체、CD4+T세포CD40배체(CD40L)표체급세포주기정황,ELISA검측각조세포배양상청중Th1형세포인자IFN-γ급Th2형세포인자IL-4수평.결과 HK-2세포경전염pcDNA3.1-myc-HBx질립후,가고표체HBx.전염HBx기인후HK-2세포CD40표체현저상조(P＜0.01)；여대조조상비,실험조CD4+T세포표면CD40L표체야현저증가(P＜0.01),S기여G2/M기세포수지화증다(P＜0.01),배양상청중IFN-γ수평승고(P＜0.05),IL-4수평강저(P＜0.05).결론 신소관상피세포경HBx전염후,기공자격분자표체상조,병촉진CD4+T세포증식,유도CD4+T세포향Th1방향분화.유차추측,을간상관성신염신조직면역손상소도치병변적지속진전야가능여차인소적삼여상관.
    Objective To investigate the effect of renal tubular epithelial cells with hepatitis B virus x (HBx) gene on mediating T-cell activation and differentiation.Methods The eukaryotic vector pcDNA3.1-myc-HBx containing HBx gene was transiently transfected into HK-2 cells by lipofectamine mediation.The expression of HBx was confirmed by real-time PCR and Western blotting.CD4+ T cell in peripheral blood of health volunteer was isolated and purified by immunomagnetic beads.Subsequently,purified CD4+T cells were co-cultured with HK-2 cells.Flow cytometry was used to detecte the expression of CD40 of HK-2 and the expression of CD40L and cell cycle of CD4+ T cells.ELISA assay was used to quantify the IFN-γ and IL-4 in co-culture supernatant.The experiment was divided into four groups:experimental group(HK-2 cells with HBx + CD4 +T cells),negative control group (HK-2 cells with pcDNA3.1-myc +CD4+T cells),blank control group (HK-2 cells +CD4+T cells) and separate culture group (CD4+T cells).Results Real-time PCR and Western blotting results verified that HBx was successfully expressed in HK-2 cells after transfection.After transfection of HBx gene,the expression of CD40 was significantly increased on HK-2 cells(P ＜ 0.01).Compared with control groups,the expression level of CD40L and the percentage of cells at S and G2/M phase increased(all P ＜ 0.01).Meanwhile,the level of IFN-γ in the supernatant was higher(P ＜ 0.05),but the level of IL-4 was lower in experimental group(P ＜ 0.05).Conclusions Over-expression of HBx gene may up-regulate the expression of costimulatory molecules CD40 of renal tubular epithelial cells,which may active CD4+ T cells,promote the proliferation of CD4+ T ceils and up-regulate Th1-type cytokines.These events may induce immune injury of renal in hepatitis B virus-associated glomerulonephritis.
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