CAJ | 학술논문

目的：观察沉默调节蛋白1（Sirt1）过表达对转化生长因子（TGF）-β1诱导人肾小管上皮细胞凋亡的影响及其机制研究。方法将体外培养人肾小管上皮细胞分为正常对照组、TGF-β1刺激组及TGF-β1＋Sirt1过表达组。采用原位末端转移酶标记技术（TUNEL）检测细胞凋亡；流式细胞术及hoechst33258染色法分别检测细胞凋亡百分比；蛋白质印迹法检测Sirt1、结缔组织生长因子（CTGF）、Bax、Bcl-2的表达；实时荧光定量聚合酶链反应（Real time-PCR）检测Sirt1、CTGF、Bax、Bcl-2mRNA水平；活性比色法检测Sirt1活性；免疫细胞化学方法检测Sirt1表达。结果与正常对照组相比，TGF-β1刺激组肾小管上皮细胞Sirt1表达及活性均显著下降，同时CTGFmRNA及蛋白水平显著上升，细胞凋亡百分比明显增加（P＜0．01），Bax/Bcl-2比率明显升高（P＜0．05）。Sirt1过表达能够显著抑制TGF-β1诱导的CTGF水平升高，降低肾小管上皮细胞凋亡百分比，减少Bax/Bcl-2比率（P＜0．05）。结论 Sirt1过表达能够抑制TGF-β1诱导的人肾小管上皮细胞凋亡，并且该作用的实现可能是部分通过抑制CTGF实现的。
목적：관찰침묵조절단백1（Sirt1）과표체대전화생장인자（TGF）-β1유도인신소관상피세포조망적영향급기궤제연구。방법장체외배양인신소관상피세포분위정상대조조、TGF-β1자격조급TGF-β1＋Sirt1과표체조。채용원위말단전이매표기기술（TUNEL）검측세포조망；류식세포술급hoechst33258염색법분별검측세포조망백분비；단백질인적법검측Sirt1、결체조직생장인자（CTGF）、Bax、Bcl-2적표체；실시형광정량취합매련반응（Real time-PCR）검측Sirt1、CTGF、Bax、Bcl-2mRNA수평；활성비색법검측Sirt1활성；면역세포화학방법검측Sirt1표체。결과여정상대조조상비，TGF-β1자격조신소관상피세포Sirt1표체급활성균현저하강，동시CTGFmRNA급단백수평현저상승，세포조망백분비명현증가（P＜0．01），Bax/Bcl-2비솔명현승고（P＜0．05）。Sirt1과표체능구현저억제TGF-β1유도적CTGF수평승고，강저신소관상피세포조망백분비，감소Bax/Bcl-2비솔（P＜0．05）。결론 Sirt1과표체능구억제TGF-β1유도적인신소관상피세포조망，병차해작용적실현가능시부분통과억제CTGF실현적。
Objective To investigate the effect of Sirt1 overexpression on TGF-β1-induced apoptosis in human kidney proximal tubular cell line (HK-2) and its mechanism. Methods Cultured HK-2 cells were divided into normal group (NG), TGF-β1(5 ng/ml) group, TGF-β1(5 ng/ml) +Sirt1 overexpression plasmid group. Apoptosis of HK-2 cells was analyzed by DeadEndTM Fluorometric TUNEL System. Meanwhile, apoptotic cell percentage was also observed by flow cytometry of Annexin V/PI staining assay and hoechst 33258 staining. The expression levels of Sirt1, CTGF, Bax, Bcl-2 were observed by western blot. The expression levels of Sirt1, CTGF, Bax, Bcl-2 mRNA were observed by real time-PCR. Sirt1deacetylase activity was colorimetrically measured by the Sirt1 deacetylase activity assay kit. Immuno-cytochemistry was used to investigate the location and level of expression of Sirt1 protein. Results Compared with NG, the expression of CTGF (P<0.01), the number of cell apoptosis (P<0.01) and ratio of Bax/Bcl-2 (P<0.05) signifi-cantly increased,while the expression and activity of Sirt1 decreased dramatically in HK-2 cells in the TGF-β1 group (P<0.01). Transfection with Sirt1 overexpression plasmid ameliorated TGF-β1-induced increase in CTGF, cell apopto-sis, ratio of Bax/Bcl-2 increase in HK-2 cells (P<0.05). Conclusion Sirt1 overexpression ameliorates TGF-β1-induced human kidney proximal tubular cell line apoptosis by inhibiting CTGF.