中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2009年
7期
503-508
,共6页
方展%孙希锋%何方方%朱忠华%张春
方展%孫希鋒%何方方%硃忠華%張春
방전%손희봉%하방방%주충화%장춘
足细胞%醛固酮%基质金属蛋白酶类%肾小球基底膜
足細胞%醛固酮%基質金屬蛋白酶類%腎小毬基底膜
족세포%철고동%기질금속단백매류%신소구기저막
Podocytes%Aldosterone%Matrix metalloproteinases%Glomemlar basement membrane
目的 观察醛同酮(ALD)刺激对足细胞培养上清液中基质金属蛋白酶2、9(MMP-2、MMP-9)活性、Ⅳ型胶原的影响及探讨ALD对足细胞细胞外基质分泌、降解的调节机制.方法 分别用不同浓度ALD(10-11、10-9、10-7mol/L)以不同时间(24、48、72 h)作用足细胞,并设立空白对照组.用明胶酶谱、Western印迹、ELISA方法检测培养上清液中MMP-2、MMP-9、Ⅳ型胶原α5链及TGF-β1;流式细胞仪检测足细胞黏附率,同时观察ALD受体拮抗剂螺内酯(SPI)及TGF-β1受体抑制剂对上述效应的阻断作用.结果 与对照组相比,ALD以时间及剂量依赖性导致培养上清液中MMP-2、MMP-9活性升高(P<0.05);Ⅳ型胶原α5链蛋白表达下降(P<0.05);TGF-β1蛋白表达升高(P<0.05).SPI可完全阻断,而TGF-β1受体抑制剂SB431542可部分阻断ALD刺激足细胞引起的MMP-2、MMP-9活性升高、Ⅳ型胶原α5链蛋白及足细胞黏附率的下降(P<0.05).结论 ALD通过TGF-β1途径使足细胞MMP-2、MMP-9活性升高,Ⅳ型胶原α5链蛋白表达下降,足细胞黏附率下降,从而使足细胞分泌基底膜成分异常,基底膜合成及降解失衡,导致足细胞损伤.
目的 觀察醛同酮(ALD)刺激對足細胞培養上清液中基質金屬蛋白酶2、9(MMP-2、MMP-9)活性、Ⅳ型膠原的影響及探討ALD對足細胞細胞外基質分泌、降解的調節機製.方法 分彆用不同濃度ALD(10-11、10-9、10-7mol/L)以不同時間(24、48、72 h)作用足細胞,併設立空白對照組.用明膠酶譜、Western印跡、ELISA方法檢測培養上清液中MMP-2、MMP-9、Ⅳ型膠原α5鏈及TGF-β1;流式細胞儀檢測足細胞黏附率,同時觀察ALD受體拮抗劑螺內酯(SPI)及TGF-β1受體抑製劑對上述效應的阻斷作用.結果 與對照組相比,ALD以時間及劑量依賴性導緻培養上清液中MMP-2、MMP-9活性升高(P<0.05);Ⅳ型膠原α5鏈蛋白錶達下降(P<0.05);TGF-β1蛋白錶達升高(P<0.05).SPI可完全阻斷,而TGF-β1受體抑製劑SB431542可部分阻斷ALD刺激足細胞引起的MMP-2、MMP-9活性升高、Ⅳ型膠原α5鏈蛋白及足細胞黏附率的下降(P<0.05).結論 ALD通過TGF-β1途徑使足細胞MMP-2、MMP-9活性升高,Ⅳ型膠原α5鏈蛋白錶達下降,足細胞黏附率下降,從而使足細胞分泌基底膜成分異常,基底膜閤成及降解失衡,導緻足細胞損傷.
목적 관찰철동동(ALD)자격대족세포배양상청액중기질금속단백매2、9(MMP-2、MMP-9)활성、Ⅳ형효원적영향급탐토ALD대족세포세포외기질분비、강해적조절궤제.방법 분별용불동농도ALD(10-11、10-9、10-7mol/L)이불동시간(24、48、72 h)작용족세포,병설립공백대조조.용명효매보、Western인적、ELISA방법검측배양상청액중MMP-2、MMP-9、Ⅳ형효원α5련급TGF-β1;류식세포의검측족세포점부솔,동시관찰ALD수체길항제라내지(SPI)급TGF-β1수체억제제대상술효응적조단작용.결과 여대조조상비,ALD이시간급제량의뢰성도치배양상청액중MMP-2、MMP-9활성승고(P<0.05);Ⅳ형효원α5련단백표체하강(P<0.05);TGF-β1단백표체승고(P<0.05).SPI가완전조단,이TGF-β1수체억제제SB431542가부분조단ALD자격족세포인기적MMP-2、MMP-9활성승고、Ⅳ형효원α5련단백급족세포점부솔적하강(P<0.05).결론 ALD통과TGF-β1도경사족세포MMP-2、MMP-9활성승고,Ⅳ형효원α5련단백표체하강,족세포점부솔하강,종이사족세포분비기저막성분이상,기저막합성급강해실형,도치족세포손상.
objective To assess the effect of aldosterone on the production of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9)and collagen Ⅳ in culture supematants of podocytes and the possible molecular mechanisms involved in the influence of aldosterone on the synthesis and degradation of extracellular matrix produced by podocytes. Methods Podecytes were treated with aldosterone at the concentration of 10-11, 10-9, 10-7 mol/L respectively. Cultured podocytes were examined at 24, 48 and 72 hours respectively. Spironolactone, a receptor antagonist of aldosterone, was added to observe the blocking effect on aldosterone. An inhibitor of TGF-β1 receptor was used to determine whether the effect of aldosterone on podocytes were mediated through TGF-β1 system. The enzymatic activities of MMP-2 and MMP-9 were assayed by gehtin zymography. Collagen Ⅳ 0.5 chain and TGF-β1 proteins released into culture supematants were assessed by Western blot and ELISA analysis. The adhesion rate of podocytes was monitored by flow cytometry. Results Aldosterone increased the activities of MMP-2 and MMP-9 in a dose- and time-dependent manner (P<0.05). Aldosterone decreased the level of collagen Ⅳ or5 chain protein in culture supernatants (P<0.05). Meanwhile, the expression of TGF-β1 was also increased (P<0.05). Spironolactone completely abolished the above-mentioned changes(P< 0.05). Blockage of TGF-β1 signaling with SB431542 prevented the aldosterone-induced upregulation of MMP-2 and MMP-9 as well as the downregulation of the collagen Ⅳ α5 chain protein and the adhesion rate of podocytes (P<0.05). Conclusions Aldosterone increases the activities of MMP-2 and MMP-9 but decreases the expression of collagen Ⅳ α5 chain and the adhension rate of podocytes possibly via TGF-β1 signaling pathway. Such alterations may contribute to glomerular podocyte injury associated with the GBM abnormality caused by the imbalance between matrix synthesis and degradation.
