脂蛋白脂酶过表达促进极低密度脂蛋白诱导人系膜细胞的胞内脂质积聚和单核细胞趋化蛋白1表达

Overexpression of lipoprotein lipase in mesangial cells enhances VLDL-induced cellular lipid accumulation and MCP-1 secretion

目的通过在人肾小球系膜细胞转染脂蛋白脂酶（LPL）基因，观察LPL在系膜细胞摄取极低密度脂蛋白（VLDL）中的作用并对其介导肾损害的可能机制进行探讨。方法以携带野生型人LPL基因（LPLwt）、无活性的突变型人LPL基因（LPL194）和对照人碱性磷酸酶基因（AP）的重组腺病毒转染人肾小球系膜细胞（HMCL）[Ad—LPLwt（活性刑）、Ad—LPL194（无活性型）和Ad-AP（对照病毒）]，RT—PCR检测细胞JPL mRNA表达；细胞免疫化学染色检测细胞LPL蛋白表达；放射性核素标记的脂质体底物法检测LPL活性。分别以油红“O”染色和酶法定性和定量检测VLDL诱导的细胞内脂质沉积；MTI法检测VLDL刺激的HMCL增殖；实时荧光定量RT—PCR和ELISA法柃测HMCL单核细胞趋化蛋白1（MCP-1）mRNA和蛋白的表达水平；HMCL与T-H蛋白1（THP-1）单核细胞的共孵育，检测单核细胞向系膜细胞的趋化。结果细胞内脂质沉积分析显示，与Ad—AP组相比，Ad—LPLwt组和Ad—LPL194组细胞内三酰甘油的积聚分别增加3．55倍（P〈0．01）和0．52倍（P〈0．05）。细胞增殖实验表明，在40mg／LVLDL刺激下，Ad－LPLwt组细胞上清较Ad—AP组对HMCL有更强的促增殖作用（0．2282±0．0168比0．1805±0．0254，P〈0．05）。与Ad—AP组相比，Ad—LPLwt组细胞MCP－1mRNA表达增加0．39倍（P〈0．05），蛋白表达上调1．18倍（P〈0．01）。Ad—LPLwt组THP－1单核细胞向系膜细胞的趋化能力也最强，为Ad—AP组的1．69倍（P〈0．05）。结论活性型和非活性型LPL均能促进VLDL诱导的系膜细胞内三酰甘油的积聚，且活性型LPL起主要作用。在商VLDL条件下，LPL促进人系膜细胞转变为泡沫细胞，扩大VLI）L对系膜细胞的促增殖效应，上调系膜细胞MCP-1的分泌及增加对THP-1细胞的趋化。LPL可能作为一个重要的因素参与富含三酰甘油的脂蛋白介导的肾损害的发生和发展。

Objective To investigate the role of LPL in enhancing VLDL uptake in mesangial cells and modulating VLDL-mesangial interaction. Methods Human wild type LPL （LPLwt）, catalytically inactive LPL （LPL194） or control alkaline phosphatase （AP） were expressed in human mesangial cell line （HMCL） via adenoviral vectors. The expression of LPL mRNA and protein was detected by RT-PCR and innnunochemistry staining, respectively. LPL activity was assayed by radioisotope labeled liposome substrate. Cellular lipid deposition was visualized by oil red O staining and analyzed quantitatively by standard enzymatic procedures. Effect of LPL on HMCL proliferation was evaluated by colorimetric assay using MTT. MCP-1 mRNA and protein levels in treated HMCLs were determined by real-time quantitative RT-PCR and enzyme-linked immunosorbent assay respectively. For adhesion study, HMCLs were treated with VLDL for six hours, followed by one-hour incubation with Tamm-Horsfall protein-1 （THP-1） cells. Results Compared with HMCLs transfected by Ad-AP, the lever of cellular triglyceride content was sharply increased in Ad-LPLwt transfeeted HMCLs [（109.11±5.01） mg/g protein vs （23.98±3.23） mg/g protein, P〈0.01] and was slightly increased in Ad-LPL194 transfected HMCLs [（36.33±2.64） mg/g protein vs （23.98±3.23） mg/g protein, P〈0.05]. LPLwt amplified VLDL-driven mesangial cells proliferation. Compared to the HMCL-Ad-AP, MCP-I mRNA and protein expression increasd by 39% （P〈0.05） and 171% （P〈0.01） in HMCL-Ad-LPLwt, and the amount of THP-1 cells adhering to HMCL-Ad-I,PLw! was increased by 1.69-fold （P〈0.05）, without significant difference between HMCL-Ad-LPL194 and HMCL-Ad-AP. Conclusions Overexpression of either active or inactive LPL in HMCLs accelerates VLDL-induced triglyceride accumulation, and enzymolysis action of LPL may be the major factor in this process. Active LPL significantly amplifies VLDL- induced proliferative effect on mesangial cells and enhances monocyte adhesion to mesangial cells through up-regulation of MCP-1. Hence, LPL may be an important contribution to initiation and progression of renal injury, mediated by triglyceride-rich lipoproteins.