贵州医科大学学报

2020, v.45;No.238(07) 745-751+759

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橙皮素对PDGF-BB诱导的大鼠原代胸主动脉VSMCs表型转化的影响
Effect of Hesperetin on Phenotypic Transition of Rat Primary Thoracic Aortic Vascular Smooth Muscle Cells Induced by PDGF-BB

李晓杰;覃瑛;周谊霞;
LI Xiaojie;QIN Ying;ZHOU Yixia;College of Nursing,Guizhou Medical University;Department of Pathology,the Affiliated Hospital of Guizhou Medical University;

摘要(Abstract):

目的:探讨橙皮素(HES)对血小板源性生长因子-BB(PDGF-BB)诱导的大鼠胸主动脉血管平滑肌细胞(VSMCs)表型转化的影响。方法:4周龄健康雄性SD大鼠1只,5%水合氯醛麻醉后,采用组织块贴壁法提取大鼠胸主动脉原代VSMCs并培养,取对数生长期细胞,采用细胞免疫荧光化学法鉴定VSMCs平滑肌肌动蛋白-α(α-SMA),采用Cell Counting Kit-8(CCK-8)试剂盒检测2、5、10、25及50μg/L PDGF-BB和10、50、100、150、200、250及300μmol/L的HES对VSMCs增殖活性的影响,筛选PDGF-BB最佳造模条件和HES最佳干预浓度;VSMCs分为对照组(0μg/L PDGF-BB)、造模组(25μg/L PDGF-BB)及加药组(25μg/L PDGF-BB+100μmol/L橙皮素),采用划痕实验检测各组VSMCs的细胞迁移能力,采用Western blot检测各组VSMCs的α-SMA、平滑肌22α(SM22α)、弹性蛋白原(ELN)及增殖细胞核抗原(PCNA)蛋白的表达。结果:原代平滑肌细胞呈长梭形,传代2 d后VSMCs有典型的"峰-谷"状特征,α-SMA鉴定阳性率> 95%; PDGF-BB最佳造模浓度是25μg/L,HES最佳干预浓度是100μmol/L;与对照组比较,造模组VSMCs迁移率增加,α-SMA及SM22α蛋白表达下降,但ELN和PCNA蛋白表达上升(P <0. 001);与造模组比较,加药组VSMCs迁移率降低,α-SMA及SM22α蛋白表达上升,但ELN、PCNA蛋白表达下降(P <0. 001)。结论:PDGF-BB可诱导大鼠胸主动脉VSMCs的增殖及增强其迁移能力,HES可通过抑制PDGF-BB的作用从而影响VSMCs的表型转化。
Objective: To investigate the effect of hesperetin on the phenotype transition of rat thoracic aortic vascular smooth muscle cells( VSMCs) induced by platelet-derived growth factor-BB( PDGF-BB). Methods: One 4-week-old healthy male SD rat was anesthetized with 5% chloral hydrate,and the primary VSMCs of rat thoracic aorta were extracted and cultured by tissue block adherence method. Primary VSMCs at logarithmic growth phase were stained using immunofluorescence chemistry to identify smooth muscle actin-α( α-SMA). CCK-8 kit was used to determine cell viability to screen the optimal concentrations of PDGF-BB( 2,5,10,25 and 50 μg/L) and hesperetin( 10,50,100,150,200,250 and 300 μmol/L). VSMCs were divided into control group( 0 μg/L PDGF-BB),model group( 25 μg/L PDGF-BB) and hesperetin group( 25 μg/L PDGF-BB + 100 μmol/L hesperetin). Wound healing assay was used to detect cell migration. Western Blot was used to detect the expression levels of α-SMA,smooth muscle 22α( SM22α),elastin( ELN) and proliferating cell nuclear antigen( PCNA). Results: The primary smooth muscle cells exhibited long fusiform. After 2 days of passage,they displayed a typical " peak-valley" feature. The α-SMA positive rate was over95% at 25 μg/L PDGF-BB. The optimal intervention concentration of hesperetin is 100 μmol/L. In model group,VSMCs migration rate was increased,and the expression levels of α-SMA and SM22αwere decreased,but ELN and PCNA were increased when compared with the control group( P <0. 001). Compared with model group,hesperetin treatment decreased VSMCs migration rate,and upregulated α-SMA and SM22α,but downregulated ELN and PCNA decreased( P < 0. 001).Conclusion: PDGF-BB induces the proliferation and VSMCs migration,while hesperetin inhibites the phenotype transition of VSMCs induced by PDGF-BB.

关键词(KeyWords): 细胞增殖;血管平滑肌细胞;原代培养;血小板源性生长因子-BB;橙皮素;表型转化
cell proliferation;vascular smooth muscle cells(VSMCs);primary culture;plateletderived growth factor-BB(PDGF-BB);hesperetin(HES);phenotypic transition

Abstract:

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基金项目(Foundation): 国家自然科学基金(81860089)

作者(Author): 李晓杰;覃瑛;周谊霞;
LI Xiaojie;QIN Ying;ZHOU Yixia;College of Nursing,Guizhou Medical University;Department of Pathology,the Affiliated Hospital of Guizhou Medical University;

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DOI: 10.19367/j.cnki.2096-8388.2020.07.001

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