目的 探讨肺瘤平膏对肺癌细胞培养上清诱导的骨髓来源内皮祖细胞(EPCs)增殖、管腔形成能力以及PI3K/AKT信号通路的影响。方法 采用密度梯度离心法分离SD大鼠骨髓单个核细胞,并进行体外诱导培养,摄取Dil-Ac-LDL与结合FITC-UEA-1双染实验、免疫荧光化学鉴定其表面抗原CD133与血管内皮生长因子受体2(VEGFR2)等方法对其进行鉴定。分别设置浓度效应组(空白对照组、肺瘤平膏高剂量组和低剂量组大鼠血清分别处理)和时间效应组(肺瘤平膏高剂量组大鼠含药血清分别处理0、12、24、48 h)。利用MTT法、Matrigel法检测EPCs增殖和管腔形成能力。应用Western blot法检测各组EPCs中NF-κB、p-Akt及PI3K p85蛋白的表达量。结果 骨髓单个核细胞培养7 d后,多呈圆形、短梭形等形态,贴壁生长,且95%以上的细胞呈Dil-Ac-LDL和FITC-UEA-1双染阳性,且同时阳性表达CD133和VEGFR2。肺瘤平膏高剂量组大鼠含药血清处理EPCs 48 h后,细胞增殖率明显低于空白对照组(P<0.01),且肺瘤平膏对EPCs增殖的抑制作用具有时间依赖性和剂量依赖性。经Matirgel实验,肺瘤平膏高剂量组大鼠含药血清作用48 h后EPCs管腔形成数量也少于空白对照组(P<0.05)。经Western blot法检测,肺瘤平膏高剂量组EPCs中NF-κB、p-Akt及PI3K p85蛋白表达量低于空白对照组(P<0.01)。结论 肺瘤平膏含药上清可抑制A549肺癌细胞培养上清诱导的骨髓源性EPCs增殖和管腔形成活性,PI3K/AKT信号通路可能在其中发挥着一定作用。
Abstract
OBJECTIVE To discuss the effect of feiliuping ointment on proliferation, angiogenesis and PI3K/AKT pathway of rat bone marrow-derived endothelial progenitor cells (EPCs) in A549 lung tumor cells supernatant. METHODS Bone marrow mononuclear cells were isolated from Sprague-Dawley rats by density gradient centrifugation methods and cultured in EGM-2 MV medium. And fluorescent immunocytochemistry was used to detect CD133 and vascular endothelial growth factor receptor 2 (VEGFR2) expression. EPCs were treated with 0.9% sodium chloride solution (blank control) or serum of rats treated with high dose and low dose Feiliuping ointment for 48 h to explore the concentration-effect, or with serum of rats treated with high dose feiliuping ointment for 0, 12, 24, 48 h to explore the time-effect. MTT assay and Matrigel method were used to detect the proliferation and angiogenesis of EPCs. The NF-κB, p-Akt, PI3K p85 proteins of EPCs were detected by Western blot. RESULTS After induced culture for 7 d, the isolated bone marrow mononuclear cells exhibited round or short spindle-shaped appearance, were Dil-Ac-LDL and FITC-UEA-1 positive, and expressed CD133+VEGFR2+. After being treated with high dose feiliuping ointment serum for 48 h, OD490 nm value was lower than that in the control group or low dose feiliuping ointment group (P<0.05). The inhibition effect of rat serum after Feiliuping ointment administration on EPCs was dose-dependent and time-dependent. Matrigel tube formation assays showed that the counts of tube formation of EPCs in high dose Feiliuping ointment group was lower than that in control group or low dose of feiliuping ointment group (P<0.05). And the levels of NF-κB, p-Akt, PI3K p85 proteins in high dose of feiliuping ointment group were lower than those in control group(P<0.01). CONCLUSIONS Treatment with administration can Feiliuping ointment serum would inhibit the proliferation and tube formation of bone marrow-derived EPCs in A549 supernatant via the down-regulation of PI3K/AKT signaling pathway.
关键词
肺瘤平膏 /
骨髓源性内皮祖细胞 /
A549细胞培养上清 /
管腔形成 /
PI3K/AKT信号通路
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Key words
feiliuping ointment /
bone marrow-derived endothelial progenitor cell /
cell supernatant /
angiogenesis /
PI3K/AKT pathway
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中图分类号：
R965
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参考文献
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脚注
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基金
国家自然科学基金项目资助(81473638);河南省科技攻关计划资助(社会发展领域)(172102310602)
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