中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2009年
9期
692-697
,共6页
杜新%黄颂敏%唐万欣%柳飞%赖学莉
杜新%黃頌敏%唐萬訢%柳飛%賴學莉
두신%황송민%당만흔%류비%뢰학리
单糖转运蛋白质类%肾小球系膜细胞%胰岛素%糖尿病肾病%Cbl相关蛋白%纤维状肌动蛋白
單糖轉運蛋白質類%腎小毬繫膜細胞%胰島素%糖尿病腎病%Cbl相關蛋白%纖維狀肌動蛋白
단당전운단백질류%신소구계막세포%이도소%당뇨병신병%Cbl상관단백%섬유상기동단백
Monosaccharide transport proteins%Mesangial cells%Insulin%Diabetic nephropathies%Cbl-associated protein%F-actin
目的 探讨高糖和胰岛素对肾小球系膜细胞(GMC)葡萄糖转运蛋白4(GLUT4)和Cbl相关蛋白(CAP)的mRNA表达及细胞骨架纤维状肌动蛋白F-actin的影响,探讨糖尿病肾病发生发展中GLUT4及其下游分子F-actin和CAP的重要作用.方法 将细胞分为8组:正常对照组、生理浓度胰岛素(10-9 mol/L)组、低浓度胰岛素(10-8 mol/L)组、高浓度胰岛素(10-6mol/L)组、高糖(30 mmol/L)组、甘露醇组(25 mmol/L 甘露醇+5 mmol/L葡萄糖)、高糖加高浓度胰岛素组、高糖加生理浓度胰岛素组.采用RT-PCR法和免疫组化法,观察不同情况下GMC中GLUT4蛋白和mRNA以及CAP mRNA的表达及其变化.Rhodamine-phalloidin染色和激光共聚焦显微镜观察F-actin形态及荧光强度.结果 正常对照组GMC中GLUT4蛋白和mRNA以及CAP mRNA有一定表达,而生理浓度胰岛素组与正常对照组差异均无统计学意义.高糖组GLUT4蛋白(P<0.01)和mRNA(P<0.05)以及CAP mRNA(P<0.01)表达均显著减少,F-actin解聚增加(P<0.01);而甘露醇组以上各指标与对照组差异均无统计学意义.低浓度胰岛素组和高浓度胰岛素组GLUT4 mRNA表达分别为生理浓度胰岛素组的2.06倍和2.66倍,GLUT4蛋白表达分别为对照组的1.93倍和2.83倍,CAP mRNA表达分别为对照组的1.91倍和2.15倍,F-actin荧光强度分别为对照组的1.296倍及1.224俯,均呈一定的浓度依赖性.高糖加高浓度胰岛素组GLUT4 mRNA表达为高糖组的2.15倍(P<0.05),GLUT4蛋白表达为高糖组的2.08倍(P<0.01),CAP mRNA表达为高糖组的2.14倍(P<0.01),F-actin荧光强度为高糖组的1.838倍(P<0.01).GI-UT4 mRNA与CAP mRNA呈正相关(r=0.905,P=0.002);GLUT4与F-actin呈止相关(r=0.929,P=0.001).结论 (1)正常GMC中GLUT4 mRNA与蛋白、CAP mRNA有一定表达.(2)高糖可抑制GLUT4的蛋白和mRNA以及CAP mRNA表达,促进F-actin解聚.(3)胰岛素能部分拮抗高糖导致系膜细胞中GLUT4的蛋白和mRNA以及CAP mRNA表达的下调作用.(4)GLUT4、CAP和F-actin是糖尿病肾病发生发展的重要影响因子之一.
目的 探討高糖和胰島素對腎小毬繫膜細胞(GMC)葡萄糖轉運蛋白4(GLUT4)和Cbl相關蛋白(CAP)的mRNA錶達及細胞骨架纖維狀肌動蛋白F-actin的影響,探討糖尿病腎病髮生髮展中GLUT4及其下遊分子F-actin和CAP的重要作用.方法 將細胞分為8組:正常對照組、生理濃度胰島素(10-9 mol/L)組、低濃度胰島素(10-8 mol/L)組、高濃度胰島素(10-6mol/L)組、高糖(30 mmol/L)組、甘露醇組(25 mmol/L 甘露醇+5 mmol/L葡萄糖)、高糖加高濃度胰島素組、高糖加生理濃度胰島素組.採用RT-PCR法和免疫組化法,觀察不同情況下GMC中GLUT4蛋白和mRNA以及CAP mRNA的錶達及其變化.Rhodamine-phalloidin染色和激光共聚焦顯微鏡觀察F-actin形態及熒光彊度.結果 正常對照組GMC中GLUT4蛋白和mRNA以及CAP mRNA有一定錶達,而生理濃度胰島素組與正常對照組差異均無統計學意義.高糖組GLUT4蛋白(P<0.01)和mRNA(P<0.05)以及CAP mRNA(P<0.01)錶達均顯著減少,F-actin解聚增加(P<0.01);而甘露醇組以上各指標與對照組差異均無統計學意義.低濃度胰島素組和高濃度胰島素組GLUT4 mRNA錶達分彆為生理濃度胰島素組的2.06倍和2.66倍,GLUT4蛋白錶達分彆為對照組的1.93倍和2.83倍,CAP mRNA錶達分彆為對照組的1.91倍和2.15倍,F-actin熒光彊度分彆為對照組的1.296倍及1.224俯,均呈一定的濃度依賴性.高糖加高濃度胰島素組GLUT4 mRNA錶達為高糖組的2.15倍(P<0.05),GLUT4蛋白錶達為高糖組的2.08倍(P<0.01),CAP mRNA錶達為高糖組的2.14倍(P<0.01),F-actin熒光彊度為高糖組的1.838倍(P<0.01).GI-UT4 mRNA與CAP mRNA呈正相關(r=0.905,P=0.002);GLUT4與F-actin呈止相關(r=0.929,P=0.001).結論 (1)正常GMC中GLUT4 mRNA與蛋白、CAP mRNA有一定錶達.(2)高糖可抑製GLUT4的蛋白和mRNA以及CAP mRNA錶達,促進F-actin解聚.(3)胰島素能部分拮抗高糖導緻繫膜細胞中GLUT4的蛋白和mRNA以及CAP mRNA錶達的下調作用.(4)GLUT4、CAP和F-actin是糖尿病腎病髮生髮展的重要影響因子之一.
목적 탐토고당화이도소대신소구계막세포(GMC)포도당전운단백4(GLUT4)화Cbl상관단백(CAP)적mRNA표체급세포골가섬유상기동단백F-actin적영향,탐토당뇨병신병발생발전중GLUT4급기하유분자F-actin화CAP적중요작용.방법 장세포분위8조:정상대조조、생리농도이도소(10-9 mol/L)조、저농도이도소(10-8 mol/L)조、고농도이도소(10-6mol/L)조、고당(30 mmol/L)조、감로순조(25 mmol/L 감로순+5 mmol/L포도당)、고당가고농도이도소조、고당가생리농도이도소조.채용RT-PCR법화면역조화법,관찰불동정황하GMC중GLUT4단백화mRNA이급CAP mRNA적표체급기변화.Rhodamine-phalloidin염색화격광공취초현미경관찰F-actin형태급형광강도.결과 정상대조조GMC중GLUT4단백화mRNA이급CAP mRNA유일정표체,이생리농도이도소조여정상대조조차이균무통계학의의.고당조GLUT4단백(P<0.01)화mRNA(P<0.05)이급CAP mRNA(P<0.01)표체균현저감소,F-actin해취증가(P<0.01);이감로순조이상각지표여대조조차이균무통계학의의.저농도이도소조화고농도이도소조GLUT4 mRNA표체분별위생리농도이도소조적2.06배화2.66배,GLUT4단백표체분별위대조조적1.93배화2.83배,CAP mRNA표체분별위대조조적1.91배화2.15배,F-actin형광강도분별위대조조적1.296배급1.224부,균정일정적농도의뢰성.고당가고농도이도소조GLUT4 mRNA표체위고당조적2.15배(P<0.05),GLUT4단백표체위고당조적2.08배(P<0.01),CAP mRNA표체위고당조적2.14배(P<0.01),F-actin형광강도위고당조적1.838배(P<0.01).GI-UT4 mRNA여CAP mRNA정정상관(r=0.905,P=0.002);GLUT4여F-actin정지상관(r=0.929,P=0.001).결론 (1)정상GMC중GLUT4 mRNA여단백、CAP mRNA유일정표체.(2)고당가억제GLUT4적단백화mRNA이급CAP mRNA표체,촉진F-actin해취.(3)이도소능부분길항고당도치계막세포중GLUT4적단백화mRNA이급CAP mRNA표체적하조작용.(4)GLUT4、CAP화F-actin시당뇨병신병발생발전적중요영향인자지일.
Objective To investigate the effects of high glucose and insulin on the expression of glucose transporter 4 (GLUT4), Cbl-associated protein (CAP) and cytoskeleton protein F-actin of glomerular mesangial cells (GMCs), in order to explore the function of GLUT4, Cbl-associated protein and F-actin in the pathogenesis and development of diabetic nephropathy (DN). Methods Cultured 1097 rat glomerular mesangial cells were divided into 8 groups: control, 10-9 mol/L insulin, 10-8 mol/L insulin, 10-6 mol/L insulin, high glucose (30 mmol/L), mannitol (25 mmol/L mannitol+5 mmol/L glucose), high glucose plus 10-6 mol/L insulin, high glucose plus 10-9 mol/L insulin. Expression of CAP mRNA and GLUT4 was measured by RT-PCR and immunohistochemistry method. F-actin was stained by rhodamine-pholloidin and the fluorescent intensity was calculated by image analysis system. Results The expression of GLUT4 mRNA and protein, CAP mRNA was found in normal giomerular mesangial cells (control), and there was no significant difference in 10-9 mol/L insulin group. The expression of GLUT4 mRNA (P<0.05) and protein (P<0.01), CAP mRNA (P<0.01) level was decreased in high glucose group compared with that of control group, but there was no significant difference in mannitol group. The expression of GLUT4 and CAP mRNA up-regulated with the increase of concentration of insulin. The expressions of GLUT4 mRNA in 10-8 mol/L insulin and 10-6 mol/L insulin groups were 2.06-fold and 2.66-fold of 10-9 mol/L insulin group, of GLUT4 protein were 1.93-fold and 2.83-fold of control, and of CAP mRNA were 1.91-fold and 2.15-fold of control, respectively. The expressions of GLUT4 mRNA, GLUT4 protein, CAP mRNA in high glucose plus insulin group were 2.15-fold, 2.08-fold, 2.14-fold of high glucose group respectively. High glucose decreased the fluorescent intensity of F-actin to 44.5% (P<0.01). 10-8 mol/L insulin and 10-6 mol/L insulin groups increased to 1.224-fold (P<0.05), 1.296-fold (P<0.01) in a concentration-dependent manner. The spearman correlation coefficient between GLUT4 and F-actin was 0.929 (P=0.001), between GLUT4 mRNA and CAP mRNA was 0.905 (P=0.002). Conclusions (1) A certain expression of GLUT4 mRNA and protein, CAP mRNA from GMC is found in normal glomerular mesangial cells. (2) High glucose can inhibit the expression of GLUT4 and CAP mRNA significantly, and facilitate the depolymerization of F-aetin. (3) Insulin can reverse down-regnlation of GLUT4 and CAP mRNA caused by high glucose. (4) GLUT4, CAP and F-actin are important factors in the development of DN.
