VEGF165真核表达载体的构建及其对白血病细胞增殖和化疗药物敏感性的影响

Construction of VEGF eukaryotic expression vector and its effects on proliferation and chemosensitivity in leukemia cells

目的:构建血管内皮细胞生长因子的真核表达载体,探讨其对白血病细胞的增殖以及化疗药物敏感性的影响。方法:应用常规基因克隆方法构建pEGFP-C1-hVEGF165真核表达载体,转染白血病细胞K562,采用CCK8法检测重组质粒对细胞增殖的影响,RT-PCR方法检测细胞血管内皮细胞生长因子(VEGF)mRNA的表达水平,ELISA方法定量检测细胞培养液和细胞内VEGF蛋白的表达水平,用流式细胞仪检测细胞周期。结果:经酶切鉴定和基因测序证实成功构建了VEGF165真核表达载体。稳定转染pEGFP-C1-hVEGF165的K562细胞较转染pEGFP-C1空质粒的K562细胞增殖加快,VEGF165 mRNA表达明显增加,细胞分泌VEGF蛋白水平上调。转染pEGFP-C1-hVEGF165还可以提高白血病细胞在化疗药物高三尖杉酯碱和氟尿嘧啶作用时的细胞存活率。结论:VEGF165真核表达载体可以上调白血病细胞K562的VEGF mRNA和VEGF蛋白的表达水平,从而促进白血病细胞的增殖,同时可以降低白血病细胞对化疗药物高三尖杉酯碱和氟尿嘧啶的敏感性。

AIM： To investigate the effects of EPA and DHA on oxidative stress of lipopolysaccharide-stimulated rat mesangial cells. METHODS： The glomerular mesangial cells （GMCs） were stimulated by lipopolysaccharide （LPS） and incubated with EPA （10 μmol/L or 100 μmol/L） and DHA （10 μmol/L or 100 μmol/L） for 24 h, 48 h and 72 h. The activity of SOD, GSH-Px and the level of MDA was measured. The protein and mRNA expressions of MCP-1 and TGF-β1 were detected by immunocytochemistry and real-time PCR method, respectively. RESULTS： The activities of SOD and GSH-Px were decreased and the concentration of MDA was increased when stimulated with LPS. EPA and DHA increased the activities of SOD and GSH-Px and decreased the concentration of MDA significantly. Meanwhile, the protein and mRNA expressions of MCP-1 and TGF-β1 stimulated by LPS were decreased. DHA was more effective than EPA at the same concentration. CONCLUSION： EPA and DHA enhance the activities of antioxidant enzymes, decrease the concentration of MDA and inhibit the expression of TGF-β1 and MCP-1, suggesting that the protective effect of EPA and DHA on kidney is related to the antioxidation and the inhibition of TGF-β1 and MCP-1 expression.