中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2013年
4期
300-304
,共5页
王轩%周益%袁伟杰%朱楠%尚明花
王軒%週益%袁偉傑%硃楠%尚明花
왕헌%주익%원위걸%주남%상명화
肝炎病毒,乙型%肾小管,近端%上皮细胞%受体,Notch1%细胞凋亡
肝炎病毒,乙型%腎小管,近耑%上皮細胞%受體,Notch1%細胞凋亡
간염병독,을형%신소관,근단%상피세포%수체,Notch1%세포조망
Hepatitis B virus%Kidney tubules,proximal%Epithelial cells%Receptor,Notch1%Apoptosis
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细胞株后对其表达Notch1受体的影响,并观察Notch1受体在HBx介导的肾小管上皮细胞增殖、凋亡中的作用.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细胞建立转染株,并应用短发夹RNA(shRNA)干扰技术沉默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Western印迹法验证HBx转染成功,检测细胞中Notch1受体的变化,四甲基偶氮唑盐(MTT)检测各组细胞的生长抑制率,流式细胞术检测各组细胞凋亡情况.结果 HK-2细胞经转染HBx基因后可高表达HBx,Notch1 shRNA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水平明显高于正常培养组及转染HBx空载质粒组,且该组细胞凋亡率亦明显高于正常培养组及转染HBx空载质粒组,差异均有统计学意义(14.94％ ±0.32％比13.86％ ±0.18％、13.67％±0.54％,均P＜0.05).转染HBx质粒+Notch1质粒组细胞凋亡率明显低于HBx质粒+Notch1空载质粒组,差异有统计学意义(10.10％ ±0.26％比14.79％ ±0.02％,P＜0.05),其细胞增殖抑制率亦低于HBx质粒+Notch1空载质粒组.而转染HBx质粒+ Notch1 shRNA沉默质粒组细胞凋亡率及细胞增殖抑制率均高于HBx质粒+Notch1 shRNA空载质粒组(均P＜0.05).结论 构建pcDNA3.1/myc-HBx质粒并转染肾小管上皮细胞可成功制备乙型肝炎病毒细胞感染模型,由此导致的肾小管上皮细胞生长抑制及凋亡增加可能与Notch1受体异常活化有关.
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細胞株後對其錶達Notch1受體的影響,併觀察Notch1受體在HBx介導的腎小管上皮細胞增殖、凋亡中的作用.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細胞建立轉染株,併應用短髮夾RNA(shRNA)榦擾技術沉默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Western印跡法驗證HBx轉染成功,檢測細胞中Notch1受體的變化,四甲基偶氮唑鹽(MTT)檢測各組細胞的生長抑製率,流式細胞術檢測各組細胞凋亡情況.結果 HK-2細胞經轉染HBx基因後可高錶達HBx,Notch1 shRNA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水平明顯高于正常培養組及轉染HBx空載質粒組,且該組細胞凋亡率亦明顯高于正常培養組及轉染HBx空載質粒組,差異均有統計學意義(14.94％ ±0.32％比13.86％ ±0.18％、13.67％±0.54％,均P＜0.05).轉染HBx質粒+Notch1質粒組細胞凋亡率明顯低于HBx質粒+Notch1空載質粒組,差異有統計學意義(10.10％ ±0.26％比14.79％ ±0.02％,P＜0.05),其細胞增殖抑製率亦低于HBx質粒+Notch1空載質粒組.而轉染HBx質粒+ Notch1 shRNA沉默質粒組細胞凋亡率及細胞增殖抑製率均高于HBx質粒+Notch1 shRNA空載質粒組(均P＜0.05).結論 構建pcDNA3.1/myc-HBx質粒併轉染腎小管上皮細胞可成功製備乙型肝炎病毒細胞感染模型,由此導緻的腎小管上皮細胞生長抑製及凋亡增加可能與Notch1受體異常活化有關.
목적 탐토을형간염병독X기인(HBx)전염인근단신소관상피세포계HK-2세포주후대기표체Notch1수체적영향,병관찰Notch1수체재HBx개도적신소관상피세포증식、조망중적작용.방법 용분자극륭적방법구건pcDNA3.1/myc-HBx질립급pcDNA3.1/myc-Notch1질립,전염HK-2세포건립전염주,병응용단발협RNA(shRNA)간우기술침묵Notch1기인표체.장세포분위7조:①정상배양조；②전염HBx공재질립조；③전염HBx질립조；④전염HBx질립+Notch1공재질립조；⑤전염HBx질립+Notch1질립조；⑥전염HBx질립+Notch1 shRNA공재질립조；⑦전염HBx질립+ Notch1 shRNA침묵질립조.실시정량PCR화Western인적법험증HBx전염성공,검측세포중Notch1수체적변화,사갑기우담서염(MTT)검측각조세포적생장억제솔,류식세포술검측각조세포조망정황.결과 HK-2세포경전염HBx기인후가고표체HBx,Notch1 shRNA재HK-2세포중적전염효솔야고체70％,기인침묵유효；전염HBx기인조세포Notch1수평명현고우정상배양조급전염HBx공재질립조,차해조세포조망솔역명현고우정상배양조급전염HBx공재질립조,차이균유통계학의의(14.94％ ±0.32％비13.86％ ±0.18％、13.67％±0.54％,균P＜0.05).전염HBx질립+Notch1질립조세포조망솔명현저우HBx질립+Notch1공재질립조,차이유통계학의의(10.10％ ±0.26％비14.79％ ±0.02％,P＜0.05),기세포증식억제솔역저우HBx질립+Notch1공재질립조.이전염HBx질립+ Notch1 shRNA침묵질립조세포조망솔급세포증식억제솔균고우HBx질립+Notch1 shRNA공재질립조(균P＜0.05).결론 구건pcDNA3.1/myc-HBx질립병전염신소관상피세포가성공제비을형간염병독세포감염모형,유차도치적신소관상피세포생장억제급조망증가가능여Notch1수체이상활화유관.
Objective To explore the expression of Notchl receptor in renal tubular epithelial cells transfected with HBx gene and elucidate its role in cell apoptosis.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 shRNA technique employed for silencing Notch1.HK-2 cells were divided into 7 groups of normal culture,pcDNA3.1/myc,HBx,HBx + pcDNA3.1/myc,HBx + Notch1 gene,HBx + shRNA and HBx + Notch1 shRNA.Real-time polymerase chain reaction (PCR) and Western blotting were used to confirm the expression of HBx and detect the expression of Notch1 receptor.Methyl thiazolyl tetrazolium (MTT) assay was used to observe the proliferation rate of HK-2 cells and flow cytometry to detect the apoptosis of HK-2 cells.Results HBx and Notch1 receptor were successfully expressed in HK-2 cells post-transfection.The transfection efficiency of shRNA was 70％.The expression of Notch1 receptor in pcDNA3.1/myc-HBx group was higher than that in the normal culture and pcDNA3.1/myc groups.And the apoptotic ratio was also higher than that of normal culture and pcDNA3.1/myc group.The difference was significant (14.94％ ± 0.32％ vs 13.86％ ± 0.18％,13.67％ ±0.54％,both P ＜ 0.05).The apoptotic ratio in the HBx + Notch1 gene group was lower than that in control group (10.10％ ±0.26％ vs 14.79％ ±0.02％,P ＜0.05).And the growth inhibition ratio of cells was also lower.But the apoptotic ratio and growth inhibition ratio were both higher in HBx + Notch1 shRNA group than those in HBx + shRNA group (both P ＜0.05).Conclusions HBx gene is successfully transfected into HK-2 cells.And its overexpression may induce apoptosis of HK-2 cells and growth inhibition by regulating Notch1 signal.
