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目的 探讨髓样分化蛋白2(MD- 2)在人脐静脉内皮细胞(HUVEC)中的表达及其在内毒素/脂多糖(LPS)激活HUVEC中的作用。 方法 分离、培养HUVEC,采用逆转录聚合酶链反应(RT- PCR)和蛋白免疫印迹(Westernblot)方法,检测LPS刺激前后HUVEC的MD- 2mRNA及其蛋白表达水平,用目的条带吸光度(A)值表示;HUVEC转染0. 5、1. 0、2. 0μgMD 2突变质粒(MD 2C95Y)及2. 0μgpEF BOS空载体质粒、2. 0μgMD 2质粒后, 观察LPS刺激对HUVEC核因子(NF)κB活性和其分泌白细胞介素(IL)8能力的影响, NF κB的测定值用A450nm表示。 结果 LPS刺激前, HU VECMD 2mRNA及蛋白有表达, LPS能明显上调其表达水平, 并呈一定的时间和剂量依赖性。LPS刺激前MD- 2蛋白A值为25 196±1 723,明显低于0. 01mg/LLPS刺激6h的A值(58 817±3 241,P<0. 01);转染MD 2C95Y能明显抑制LPS对HUVECNF- κB的激活和IL 8的分泌。 结论 M-D 2在LPS激活/损伤HUVEC效应中具有重要的地位。
Objective To investigate the expression of myeloid differentiation protein 2 (MD-2) in human umbilical vein endothelial cells (HUVECs) and its role in the activation of HUVEC by endotoxin / lipopolysaccharide (LPS). Methods HUVECs were isolated and cultured. The mRNA and protein expression of MD-2 in HUVECs before and after LPS stimulation were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The absorbance (A) HUVEC transfected with 0.5,1.0,2. 0μM MD2 mutant plasmid (MD 2C95Y) and 2. 0μgpEF BOS empty vector plasmid, 2.0μgMD 2 plasmid after LPS stimulation on the nuclear factor (NF) κB activity and its ability to secrete interleukin (IL) 8, NF κB was measured as A450 nm. Results Before LPS stimulation, HU VECMD 2 mRNA and protein were expressed. LPS could up-regulate the expression of HU VECMD 2 in a dose and time-dependent manner. The value of MD-2 protein A before LPS stimulation was 25 196 ± 1 723, which was significantly lower than that of 0.01 mg / L LLPS stimulation for 6 hours (58 817 ± 3 241, P <0.01) Activation of HUVECNF-κB and secretion of IL 8 by LPS. Conclusion M-D 2 plays an important role in LUV-activated / injured HUVEC.