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活性氧通过促进血管平滑肌细胞 (VSMC)增殖已被认为是动脉粥样硬化和高血压的病因之一。我们以前的实验显示 ,超氧阴离子生成剂LY835 83激活VSMC细胞外信号调节激酶 (ERK1 2 ) ,并呈现出两个效应峰值特征。早期效应出现在LY835 83作用后 10min ,晚期效应出现在LY835 83作用后 2h。LY835 83通过激活ERK1 2诱导VSMC增殖。我们推测 ,LY835 83作用的第一相是其进入细胞后产生超氧阴离子所致 ,因为超氧阴离子产生抑制剂Tiron只取消LY835 83的第一相作用 ;而第二相作用可能是由于LY835 83刺激VSMC释放ERK1 2激活因子所为。我们将这种因子命名为氧化应激诱导因子 (secretedoxida tivestress -inducedfactors,SOXF)。为了证实此一假设 ,我们采用SPSepharose、HeparinSepharose、PhenylSepharose、GelFiltration等一系列亲合层析和凝胶过滤技术 ,以分离获得的成分是否能激活ERK1 2为监测指标 ,从受LY835 83刺激 2h的VSMC条件培养基中分离出SOXF蛋白。然后通过毛细管色谱 (CCG)技术和ElectrosprayIonizationTandemMassSpectrometry技术分析发现热休克蛋白 90 -a (HSP90 -a)和CyclophilinB是主要的SOXF。免疫印迹分析发现 ,VSMC受LY835 83刺激后特异地分泌HSP90-a,而不是HSP90 -b。随后的实验发现 ,条件培养基中的H
Reactive oxygen species have been identified as one of the causes of atherosclerosis and hypertension by promoting the proliferation of vascular smooth muscle cells (VSMCs). Our previous experiments showed that the superoxide anion generator LY83583 activates VSMC extracellular signal-regulated kinase (ERK1 2) and exhibits two peak effect characteristics. Early effects appeared in LY835 83 after 10min, late effects appeared in LY835 83 after 2h. LY83583 induces VSMC proliferation by activating ERK1 2. We hypothesize that the first phase of LY83583 action is due to its production of superoxide anion after entering the cell because Tiron only cancels the first phase of LY83583 and the second phase may be due to LY83583 Stimulation of VSMC release ERK1 2 activator. We named this factor the secreted oxidative stress factor (SOXF). To confirm this hypothesis, we used a series of affinity chromatography and gel filtration techniques, such as SPSepharose, Heparin Sepharose, Phenyl Sepharose, Gel Filtration and so on, to isolate whether the obtained components could activate ERK1 2 as a monitoring index. From VSMCs stimulated by LY835 83 for 2 hours SOXF protein was isolated from conditioned media. Hsp90-a (HSP90-a) and CyclophilinB were then found to be the major SOXFs by capillary chromatography (CCG) and Electrospray Ionization TandemMass Spectrometry techniques. Immunoblot analysis revealed that VSMC secreted HSP90-a specifically but not HSP90-b upon stimulation with LY83583. Subsequent experiments found that conditioned medium H