碲化镉量子点对血管平滑肌细胞的损伤作用

摘要

目的 观察碲化镉量子点(CdTe QD)在体外对大鼠血管平滑肌细胞(VSMC)的损伤作用.方法CdTe QD(0.01~100 mg·L-1)与原代培养的大鼠胸/腹主动脉平滑肌细胞共孵育24 h,MTT法检测CdTe QD对细胞存活的抑制;钙黄绿素乙酰甲酯(calcein-AM)荧光染色观察CdTe QD对VSMC细胞活性的影响;DCFH-DA和JC-1染色、流式细胞术检测CdTe QD作用后细胞内活性氧(ROS)含量和线粒体膜电位的变化;流式细胞术检测细胞凋亡;Western蛋白印迹法检测BCL-2和BAX蛋白表达变化.结果 MTT结果显示,CdTe QD 0.01~100 mg·L-1作用VSMC细胞24 h,细胞存活率降低(P<0.01),24 h IC50值为25.60 mg·L-1.Calcein-AM荧光检测发现,CdTe QD 0.1~25 mg·L-1作用后,VSMC细胞活性下降.DCFH-DA染色结果显示,CdTe QD 0.1~25 mg·L-1使细胞内ROS逐渐增加(P<0.05,P<0.01);JC-1染色结果表明,VSMC线粒体膜电位呈浓度依赖性降低(r=0.903,P<0.01).Western蛋白印迹结果表明,CdTe QD诱导抗凋亡蛋白BCL-2表达显著降低(P<0.01),促凋亡蛋白BAX表达显著上升(P<0.01);流式细胞术FITC-AnnexinⅤ染色结果显示,CdTe QD 0.1~25 mg·L-1作用24 h能显著促进VSMC细胞凋亡(P<0.05,P<0.01).结论 CdTe QD可诱导VSMC细胞凋亡,其机制可能与胞内ROS含量上升和线粒体介导的凋亡通路激活相关.%OBJECTIVE To study the effect of CdTe quantum dots (QD) induced injuries in vascular smooth muscle cells (VSMCs) and the potential mechanism. METHODS The rat thoracic/abdominal aorta VSMCs were treated with 0.01-100 mg · L- 1 CdTe QD for 24 h. The cell survival of CdTe QD-treated VSMCs was detected with MTT assay. The cell viability of VSMCs was measured by calcein-AM staining. Then, the CdTe QD-treated VSMCs were labeled with DCFH-DA and JC-1 probes. The intracellular ROS and mitochondrial membrane potential (MMP) were examined by flow cytometry. The cellular apoptosis in VSMCs was also examined in a flow cytometer after being labeled with FITC-Annexin Ⅴ. Finally, the level of mitochondrial apoptosis pathways proteins including BCL-2 and BAX was observed by Western blotting. RESULTS MTT data showed that CdTe QD (0.01-100 mg·L-1) inhibited the cell survival (r=0.957, P<0.01), and that IC50 was 25.6 mg · L-1 at 24 h. The results of calcein-AM staining showed that the cell viability was gradually decreased. DCFH-DA staining results showed that 1-25 mg·L-1 CdTe QD induced an increase in intracellular ROS levels (P<0.05, P<0.01). The results of JC-1 data showed a concentration-dependent disruption of MMP (r=0.903, P<0.01). Moreover, the results of Western blotting suggested that CdTe QD significantly increased the expression of BCL-2 and decrease the expression of BAX in VSMCs (P<0.01). The flow cytometry results from FITC-AnnexinⅤassay showed 0.1-25 mg · L-1 CdTe QD significantly increased the apoptosis of VSMCS. CONCULSION CdTe QD induces apoptotic cell death in VSMCs, which is possibly related to the up-regulation of intra?cellular ROS and activation of mitochondria-mediated apoptosis pathways.