CAJ | 학술논문

目的：研究叉头状转录因子 O1(FoxO1)对高糖培养下大鼠肾小球系膜细胞增殖的影响及其作用机制。方法构建含组成性激活突变型 FoxO1(CA-FoxO1)编码序列的过表达慢病毒载体(LV-CA-FoxO1)及表达针对 FoxO1的小干扰 RNA 序列(siRNA-FoxO1)的慢病毒载体(LV-siRNA-FoxO1)用于上调或下调 FoxO1的表达,并构建空慢病毒载体(LV-NC-FoxO1)作为阴性对照。将系膜细胞分为5组处理,正常糖(5.6 mmol/ L,NG 组)、单纯高糖(30 mmol/ L,HG0组)、LV-NC-FoxO1+高糖(HG1组)、LV-siRNA-FoxO1+高糖(HG2组)及 LV-CA-FoxO1+高糖(HG3组)。各组细胞培养72 h 后,用 MTT 法分析系膜细胞增殖情况；流式细胞仪检测系膜细胞周期分布；实时荧光定量 PCR 及蛋白免疫印迹法分别检测 FoxO1、细胞周期蛋白依赖性激酶抑制剂 p27、细胞周期蛋白 D1(cyclin D1)及周期蛋白依赖性激酶4(CDK4)的表达情况。结果与 NG 组相比,HG0组的高糖刺激系膜细胞过度增殖；HG0组与 HG1组系膜细胞的增殖速率及FoxO1表达量均无统计学差异；LV-CA-FoxO1上调 HG2组中 FoxO1的表达(P<0.05),导致细胞周期阻滞于 G1期且抑制细胞增殖速率,并伴随着 p27表达的升高,cyclin D1与 CDK4表达的降低(均 P<0.05)；LV-siRNA-FoxO1下调 HG3组中 FoxO1的表达(P<0.05),导致细胞增殖速率加快,并伴随着 p27表达的降低,cyclin D1与 CDK4表达的升高(均 P<0.05)。结论利用转基因技术靶向性地调节 FoxO1的表达,从而通过 FoxO1/ p27信号通路调节系膜细胞的增殖。这些发现表明 FoxO1可能成为治疗早期糖尿病肾病的靶点。
목적：연구차두상전록인자 O1(FoxO1)대고당배양하대서신소구계막세포증식적영향급기작용궤제。방법구건함조성성격활돌변형 FoxO1(CA-FoxO1)편마서렬적과표체만병독재체(LV-CA-FoxO1)급표체침대 FoxO1적소간우 RNA 서렬(siRNA-FoxO1)적만병독재체(LV-siRNA-FoxO1)용우상조혹하조 FoxO1적표체,병구건공만병독재체(LV-NC-FoxO1)작위음성대조。장계막세포분위5조처리,정상당(5.6 mmol/ L,NG 조)、단순고당(30 mmol/ L,HG0조)、LV-NC-FoxO1+고당(HG1조)、LV-siRNA-FoxO1+고당(HG2조)급 LV-CA-FoxO1+고당(HG3조)。각조세포배양72 h 후,용 MTT 법분석계막세포증식정황；류식세포의검측계막세포주기분포；실시형광정량 PCR 급단백면역인적법분별검측 FoxO1、세포주기단백의뢰성격매억제제 p27、세포주기단백 D1(cyclin D1)급주기단백의뢰성격매4(CDK4)적표체정황。결과여 NG 조상비,HG0조적고당자격계막세포과도증식；HG0조여 HG1조계막세포적증식속솔급FoxO1표체량균무통계학차이；LV-CA-FoxO1상조 HG2조중 FoxO1적표체(P<0.05),도치세포주기조체우 G1기차억제세포증식속솔,병반수착 p27표체적승고,cyclin D1여 CDK4표체적강저(균 P<0.05)；LV-siRNA-FoxO1하조 HG3조중 FoxO1적표체(P<0.05),도치세포증식속솔가쾌,병반수착 p27표체적강저,cyclin D1여 CDK4표체적승고(균 P<0.05)。결론이용전기인기술파향성지조절 FoxO1적표체,종이통과 FoxO1/ p27신호통로조절계막세포적증식。저사발현표명 FoxO1가능성위치료조기당뇨병신병적파점。
Objective To study the effect and mechanism of forkhead transcriptionfactor O1( FoxO1) on proliferation of rat mesangial cells(MCs) cultured under high glucose conditions. Methods Constructing lentiviral vectors of LV-CA-FoxO1 and LV-siRNA-FoxO1 were used to upregulate or downregulate FoxO1. Moreover, negative control LV-NC-FoxO1 was also constructed. Rat MCs were separately cultured in normal glucose(5. 6 mmol/ L, NG group), only high glucose(30 mmol/ L, HG0 group), LV-NC-FoxO1 with HG(HG1 group),LV-CA-FoxO1 with HG (HG2 group), and LV-siRNA-FoxO1 with HG(HG3 group) for 72 h. MTT assay and flow cytometrywas were used to analyze cell proliferation and cell cycle distribution. The expression of FoxO1, cyclin-dependent kinase inhibitor (p27), cyclinD1, and cyclin-dependent kinase 4( CDK4) were detected by QRT-PCR and Western blot. Results The MCs proliferation rate in HG0 group was faster than that in NG group. Besides, there were no statistical differences in FoxO1 expression and proliferation rate of MCs between HG0 group and HG1 group. Nevertheless, LV-CA-FoxO1 promoted cell cycle arrest at the G1 phase and attenuated proliferation rate, along with upregulation of FoxO1 and p27 and downregulation of cyclin D1 and CDK4 in HG2 group ( all P < 0. 05). Moreover, degradation of FoxO1 by LV-siRNA-FoxO1 stimulated hyperproliferation of MCs, associating with decline of p27 and increase of cyclin D1 and CDK4 in HG3 group(all P<0. 05). Conclusion The proliferation of MCs induced by high glucose is regulated by utilizing transgenic technology targeted and regulated FoxO1 expression and consequently through FoxO1 / p27 signaling pathway. These findings indicate that FoxO1 seems to be a new therapeutic target for early diabetic nephropathy.