纤溶酶原激活物抑制物-1基因转导对肾小球系膜细胞外基质积聚的影响  

Effect of plasminogen activator inhibitor-1 gene transfer into mesangial cells on Extracellular matrix accumulation

作　　者：于志恒[1] 陈香美[1] 廖洪军[1] 叶一舟[1] 傅博[1] 

机构地区：[1]解放军总医院肾科

出　　处：《中华内科杂志》1999年第8期541-545,共5页Chinese Journal of Internal Medicine

基　　金：国家自然科学基金!3 942 10 0 5

摘　　要：目的　利用体外基因转染技术使纤溶酶原激活物抑制物 1(PAI 1)基因过表达 ,直接观察局部PAI 1对大鼠系膜细胞外基质 (ECM)积聚的影响 ,解析ECM积聚的分子机制。方法　以绿色荧光蛋白 (GFP)为报告基因 ,构建PAI 1/GFP融合基因真核表达载体 ,利用脂质体将外源PAI 1cDNA导入体外培养的大鼠系膜细胞。在活细胞状态下 ,动态观察外源基因的表达。用Northern杂交、ELISA方法检测大鼠系膜细胞纤维连接蛋白 (FN)、层连蛋白 (LN)及IV型胶原表达。结果　PAI 1基因转染后 ,转染组大鼠系膜细胞中的培养上清PAI活性明显增高 ,到 2 4小时达最高值 ( 8.16± 0 .62 )IU/ml,与对照组 ( 2 .2 7± 0 .19)IU/ml相比 ,差异有显著性 (P <0 .0 5 )。同时基因转染组FN( 0 .5 1±0 0 3 )、LN( 1.2 6± 0 .0 7)及Ⅳ型胶原 ( 0 .98± 0 .0 8)水平均较对照组明显增高 (P <0 .0 5 )。结论　首次建立了系膜细胞PAI 1的过度表达体系 ,证实细胞过度表达PAI 1可以直接导致ECM的积聚。局部PA/PAI活性的变化可能是调节系膜细胞ECM积聚与降解的重要因素。Objective To observe the influence of plasminogen activator inhibitor 1 (PAI 1) overexpression on extracellular matrix (ECM) accumulation of rat mesangial cells. Methods PAI 1/GFP green fluorescent protein fusion gene eukaryotic expression vector pCMX PAI 1 GFP was constructed and delivered into cultured rat mesangial cells by liposome. PAI 1/GFP expression was observed by fluorescent microscope. FN, LN and Type IV collagen levels were measured by Northern blot analysis and ELISA method. Results Northern blot analysis, SDS PAGE analysis and Western blot analysis demonstrated that PAI 1/GFP gene can be transcripted and translated in rat mesangial cells. PAI 1/GFP gene can express a specific protein. The recombinant PAI 1/GFP had significant inhibition activity on urokinase type plasminogen activator and displayed autonomous fluorescence. The PA activity of cell supernatants decreased markedly in gene transfer group. FN, LN and Type Ⅳ collagen levels were higher than in a control group. Conclusion Over expression of PAI 1 gene leads to mesangial ECM accumulation. The changes of PA/PAI 1 activity in supernatants of cultured rat mesangial cells might be the key factor in ECM accumulation regulation.

关 键 词：基因表达 肾小球膜 肾疾病 PAI-1 病理 

分 类 号：R692.02[医药卫生—泌尿科学]