A possible treatment strategy for cardiovascular remodeling by metabolic syndrome-The gene therapy for use of MCP-1 7ND mutated gene-

MCP-17ND：Fc嵌合体cDNA(7ND)的制备及其显性-负性活性的验证：首先，我们用聚合酶链式反应的方法，将人MCP-1氨基端缺失的突变体与人免疫球蛋白Fc链偶联，获得了7ND的cDNA。通过在293细胞中过表达7ND基因获得重组7ND蛋白，并验证了重组7ND蛋白对MCP-1诱导的巨噬细胞产生干扰素-γ的显性负活性。在WKY大鼠和SD大鼠中，7ND基因转移在肌肉中表达了7ND蛋白，ELISA法证明循环7ND水平显著升高，注射部位肌肉有明显的损伤(高达1g质粒/kg体重)。然而，我们没有检测到7ND(1g/kg)处理的OLEFT大鼠循环中7ND水平的显著表达。一种解释是模型的不同。另一个是转基因部位的肌肉损伤。因为OLEFT大鼠比WKY和SD…大得多大鼠越多，质粒液的净剂量越大，导致心肌细胞溶解、坏死。因此，我们现在正在努力寻找更有效的载体。多危险因素模型心脏重构的评估(OLETF大鼠)：我们使用OLETF大鼠来研究糖尿病和高脂状态下的心血管重构。以LETF大鼠为对照。我们用抗单核细胞趋化蛋白-1(MCP-1)的中和抗体(NAB)来替代7ND基因治疗，以阻断MCP-1的活性。从30周龄开始，给OLETF和LETF大鼠每日注射NAB或对照免疫球蛋白。在40周龄时，我们评估了心肌肥大、纤维化和心功能。与对照组相比，OLETF组大鼠心肌间质纤维化，尤其是修复性心肌纤维化和主要血管的硬化性改变明显加重。在心肌纤维化前，对照组OLETF大鼠血管周围可见巨噬细胞浸润和成纤维细胞增殖。这一现象表明，高血糖参与了炎症变化和组织损伤重塑。NAB治疗不能减少OLETF大鼠心脏巨噬细胞聚集和心肌纤维化。我们推测，NAB的抑制作用不会持续10周以下

Production of the MCP-17ND:Fc chimera cDNA (7ND) and verification of its dominant-negative activit :At first, we use PCR method to create 7ND cDNA by conjugating the amino-terminal deleted mutant of human MCP-1 and the human immunoglobulin Fc chain. Recombinant 7ND protein was obtained by overexpressing 7ND cDNA in 293 cells and we verified the dominant-negative activity of the recombinant 7ND protein on the MCP-1-induced macrophage production of interferon-gamma. In WKY rats and SD rats, 7ND gene transfer expressed 7ND protein in the muscle and significant elevation of circulating 7ND levels was documented by Elisa, having significant musclular damage at the site of injection (up to 1 g plasmid/kg body weight). However, we could not detect significant expression in circulating 7ND levels in 7ND (1 g/kg)-treated OLEFT rats. One explanation is the difference of the model. Another one is the muscle damage at the transfected site. Because OLEFT rats grow substantial larger than WKY and SD … More rats, the net dose of plasmid solution got much larger, resulting in the myocyte lysis and necrosis. Thus, we are now trying to search more efficient vectors.Evaluation of cardiac remodeling at multiple risk factor model (OLETF Rat) :We used OLETF rats to investigate cardiovascular remodeling in diabetes and hyperlipidemia status. LETF rats were use as controls. In stead of 7ND gene therapy, we used a neutralizing antibody against MCP-1 (NAb) to block MCP-1 activity. NAb or control IgG was administered everyday to OLETF and LETF rats from 30 weeks-old. At 40 weeks of age, we evaluated cardiac hypertrophy, fibrosis and cardiac function. In control OLETF rats had been greater interstitial myocardial fibrosis, especially reparative cardiac fibrosis, and sclerotic change of major vessels, compared with control LETF rats. Preceding cardiac fibrosis, macrophage infiltration and fibroblast proliferation were observed in perivascular space in control OLETF rats. This phenomenon suggested that hyperglycemia is involved in both inflammation changes and tissue damage remodeling. NAb treatment failed to reduce macrophage accumulation and cardiac fibrosis in OLETF rat hearts. We speculated that the inhibitory effects of NAb did not last for 10 weeks Less