中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2010年
12期
892-897
,共6页
相虹%黄松明%冯泉城%袁杨刚%张爱华
相虹%黃鬆明%馮泉城%袁楊剛%張愛華
상홍%황송명%풍천성%원양강%장애화
尿酸%肾小球系膜细胞%活性氧%细胞外信号调节激酶%细胞增殖
尿痠%腎小毬繫膜細胞%活性氧%細胞外信號調節激酶%細胞增殖
뇨산%신소구계막세포%활성양%세포외신호조절격매%세포증식
Uric acid%Glomerular mesangial cells%Reactive oxygen species%Extracellular signal-regulated kinases%Cell Proliferation
目的 探讨尿酸(UA)对大鼠肾小球系膜细胞(GMC)增殖的影响及其可能的机制.方法 体外培养大鼠GMC,应用不同浓度的UA(50、100、300μmol/L)刺激或应用细胞外信号调节激酶(ERK1/2)特异性抑制剂U0126(10 μmol/L)、NADPH氧化酶特异性抑制剂夹竹桃麻素(500 μmol/L)、线粒体复合体Ⅰ抑制剂鱼藤酮(10 μmol/L)预处理30 min后,再加入UA(300 μmol/L).于实验终点收集细胞,应用3H-TdR掺入法、细胞计数及流式细胞术测定GMC增殖和细胞周期变化;应用实时定量PCR、Western印迹法检测细胞周期素cyclin D1和cyclin A2的表达及ERK1/2的磷酸化水平;应用荧光探针2,7-二氯二氢荧光素乙酰乙酸(DCFDA)检测细胞内活性氧(ROS)的变化.结果 (1)与对照组相比,3H-TdR掺入法和细胞计数均显示,UA呈剂量依赖性促进GMC增殖,300 μmol/L UA刺激组其细胞数为对照组的1.5倍以上.(2)流式细胞术显示,UA呈剂量依赖性减少G1/G0期细胞数,增加S期细胞数,300 μmol/L UA刺激组其S期细胞数为对照组的2倍以上.(3)UA呈剂量依赖性促进系膜细胞周期蛋白cyclin D1和cyclin A2的表达.(4)UA呈剂量依赖性促进系膜细胞ERK1/2磷酸化且U0126能够抑制UA诱导的GMC增殖.细胞计数和3H-TdR掺入法分别显示,U0126的抑制率分别是UA 300 μmol/L刺激组的22%和31%(均P＜0.05).(5)UA呈剂量依赖性促进ROS产生增加,夹竹桃麻素能够明显抑制UA诱导的ROS生成、ERK1/2的磷酸化和系膜细胞增殖(均P＜0.05),而鱼藤酮对其无明显影响.结论 UA可促进GMC增殖,其可能的机制为UA诱导NADPH氧化酶来源的ROS产生增加,从而激活ERK1/2信号通路,引起周期蛋白表达增加,促进GMC增殖.
目的 探討尿痠(UA)對大鼠腎小毬繫膜細胞(GMC)增殖的影響及其可能的機製.方法 體外培養大鼠GMC,應用不同濃度的UA(50、100、300μmol/L)刺激或應用細胞外信號調節激酶(ERK1/2)特異性抑製劑U0126(10 μmol/L)、NADPH氧化酶特異性抑製劑夾竹桃痳素(500 μmol/L)、線粒體複閤體Ⅰ抑製劑魚籐酮(10 μmol/L)預處理30 min後,再加入UA(300 μmol/L).于實驗終點收集細胞,應用3H-TdR摻入法、細胞計數及流式細胞術測定GMC增殖和細胞週期變化;應用實時定量PCR、Western印跡法檢測細胞週期素cyclin D1和cyclin A2的錶達及ERK1/2的燐痠化水平;應用熒光探針2,7-二氯二氫熒光素乙酰乙痠(DCFDA)檢測細胞內活性氧(ROS)的變化.結果 (1)與對照組相比,3H-TdR摻入法和細胞計數均顯示,UA呈劑量依賴性促進GMC增殖,300 μmol/L UA刺激組其細胞數為對照組的1.5倍以上.(2)流式細胞術顯示,UA呈劑量依賴性減少G1/G0期細胞數,增加S期細胞數,300 μmol/L UA刺激組其S期細胞數為對照組的2倍以上.(3)UA呈劑量依賴性促進繫膜細胞週期蛋白cyclin D1和cyclin A2的錶達.(4)UA呈劑量依賴性促進繫膜細胞ERK1/2燐痠化且U0126能夠抑製UA誘導的GMC增殖.細胞計數和3H-TdR摻入法分彆顯示,U0126的抑製率分彆是UA 300 μmol/L刺激組的22%和31%(均P＜0.05).(5)UA呈劑量依賴性促進ROS產生增加,夾竹桃痳素能夠明顯抑製UA誘導的ROS生成、ERK1/2的燐痠化和繫膜細胞增殖(均P＜0.05),而魚籐酮對其無明顯影響.結論 UA可促進GMC增殖,其可能的機製為UA誘導NADPH氧化酶來源的ROS產生增加,從而激活ERK1/2信號通路,引起週期蛋白錶達增加,促進GMC增殖.
목적 탐토뇨산(UA)대대서신소구계막세포(GMC)증식적영향급기가능적궤제.방법 체외배양대서GMC,응용불동농도적UA(50、100、300μmol/L)자격혹응용세포외신호조절격매(ERK1/2)특이성억제제U0126(10 μmol/L)、NADPH양화매특이성억제제협죽도마소(500 μmol/L)、선립체복합체Ⅰ억제제어등동(10 μmol/L)예처리30 min후,재가입UA(300 μmol/L).우실험종점수집세포,응용3H-TdR참입법、세포계수급류식세포술측정GMC증식화세포주기변화;응용실시정량PCR、Western인적법검측세포주기소cyclin D1화cyclin A2적표체급ERK1/2적린산화수평;응용형광탐침2,7-이록이경형광소을선을산(DCFDA)검측세포내활성양(ROS)적변화.결과 (1)여대조조상비,3H-TdR참입법화세포계수균현시,UA정제량의뢰성촉진GMC증식,300 μmol/L UA자격조기세포수위대조조적1.5배이상.(2)류식세포술현시,UA정제량의뢰성감소G1/G0기세포수,증가S기세포수,300 μmol/L UA자격조기S기세포수위대조조적2배이상.(3)UA정제량의뢰성촉진계막세포주기단백cyclin D1화cyclin A2적표체.(4)UA정제량의뢰성촉진계막세포ERK1/2린산화차U0126능구억제UA유도적GMC증식.세포계수화3H-TdR참입법분별현시,U0126적억제솔분별시UA 300 μmol/L자격조적22%화31%(균P＜0.05).(5)UA정제량의뢰성촉진ROS산생증가,협죽도마소능구명현억제UA유도적ROS생성、ERK1/2적린산화화계막세포증식(균P＜0.05),이어등동대기무명현영향.결론 UA가촉진GMC증식,기가능적궤제위UA유도NADPH양화매래원적ROS산생증가,종이격활ERK1/2신호통로,인기주기단백표체증가,촉진GMC증식.
Objective To explore the effect of uric acid (UA) on the rat glomerular mesangial cells (GMCs) and its possible mechanism in vitro. Methods Cultured rat GMCs were treated with various concentrations of UA (50 μmol/L, 100 μmol/L, 300 μmol/L) in the presence or absence of U0126, apocynin, rotenone. The incorporation of 3H-thymidine (3H-TdR) and cell counting were used to measure GMCs proliferation. GMCs cell-cycle was analyzed by flow cytometry. CyclinD1 and cyclin A2 expression were determined by real-time PCR and Western blotting analysis. ERK1/2 phosphorylation was detected by Western blotting. Reactive oxygen species (ROS) was measured by 2, 7-dichlorofluorescein diacetate (DCFDA) fluorescence. Results (1) Uric acid increased GMCs proliferation in dose-dependent manner compared with the control groups, as assessed by 3H-TdR incorporation and cell counting. GMCs proliferation induced by 300 μmol/L uric acid was increased by more than 1.5-fold assessed by both of the methods. (2) Uric acid decreased cell number in G1/G0 phase and increased cell number in S phase in dosedependent manner, as assessed by flow cytometry. (3) Uric acid iuduced cyclin D1 and cyclin A2 expression in dose-dependent manner. (4)Uric acid increased pospho-ERK1/2 in dose-dependent manner and ERK1/2 specific inhibitor U0126 could suppress uric acid-induced cell proliferation.The inhibition percentage of U0126 was 22% and 31% assassed by cell counting and 3H-TdR incorporation, respectively. (5) Uric acid increased ROS production in dose-dependent manner.NADPH oxidase inhibitor apocynin could also significantly inhibit uric acid-induced ROS production, ERK phosphorylation and cell proliferation. In contrast, rotenone had no effect on them.Conclusion Uric acid can stimulate rat GMCs proliferation, partially by the activation of ERK pathway via NADPH oxidase-derived ROS generation.
