QCM 监测自组装膜上心肌细胞黏附及其与心血管药物作用

目的:本试验采用石英晶体微天平(QCM)实时监测大鼠心肌细胞(H9C2)在含有细胞黏附识别多肽RGD自组装膜上的动态黏附过程及随后与两种心血管药物(一种正性肌力、另一种负性肌力)相互作用。方法:在金电极表面自组装3-巯基丙酸(MPA)单层膜,并经酰胺化共价耦合细胞黏附分子KRGD,形成对大鼠心肌细胞有特异性黏附的致密分子自组装膜。QCM以动态持续的方式实时监测MPA/RGD自组装及其不同浓度梯度H9C2细胞在自组装膜金电极上的细胞黏附过程。此外,选用20,000个H9C2细胞和正性肌力药物异丙肾上腺素、负性肌力药物维拉帕米,用QCM评估了细胞-心血管药物的相互作用。结果:与裸金电极相比,MPA/RGD修饰金电极增大了H9C2细胞黏附所引起的QCM频移(△f)与动态电阻变化(△R)响应。在所试H9C2浓度范围(5×10~4-4×10~5 cells/m L),△f与H9C2浓度呈线性关系,△R与H9C2浓度呈幂函数关系。我们用细胞粘弹性指数(CVI=△R/△f)来表征细胞的粘弹性。H9C2在异丙肾上腺素作用下,△f与△R增加、细胞-QCM表面黏附加强,细胞变硬;在维拉帕米作用下,△f与△R降低、细胞QCM表面黏附减弱,细胞变软。结论:QCM可用于不同浓度大鼠心肌细胞的动态细胞黏附监测,并可基于其细胞黏附与细胞黏弹性测定能力区分正性与负性肌力药物而可望用于心血管药物的筛选。

Real Time Monitoring of Cardiomyocytes Adhesion and Cells-Cardiovascular Drug Interactions Using RGD-Modified QCM

Objective:Quartz crystal microbalance (QCM) was used for the dynamic monitoring of cell adhesion in real time of rat myocardial cells (H9C2) onto self-assembled film containing cell recognition peptide RGD, as well as the subsequent interactions with two cardiovascular drugs (one positively inotropic and the other negatively inotropic).Methods:A self-assembling 3-mercaptopropionic acid (MPA) monolayer was formed on the gold QCMelectrode surface, allowing for subsequent covalent coupling with the cell adhesion peptide KRGD, to form a dense molecular self-assembled monolayer with specific recognition motif for myocardial cells. QCMwas successfully used for the dynamic monitoring of the MPA/RGD self-assembly process and the subsequent myocardial cell adhesion process in the concentration range of 5× 104 - 4× 105 cells/mL of H9C2 cells in real time. Additionally, QCMwas used to assess the cells-cardiovascular drug interactions using 20,000 H9C2 cells and positive inotropic drug, isoprenaline and negative inotropic drug, verapamil.Results:Compared to bare gold electrode, the MPA/RGD-modified gold electrode yielded enhanced frequency shift (Df) and motional resistance change (DR) with the adhesion of H9C2 cells. In the tested H9C2 concentration range (5× 104 - 4× 105 cells/mL), Df was linearly related to the cell concentration, whereas DR was a power function of the cell concentration. Cell Viscoelastic Index (CVI= DR/Df) was used to characterize the cells'' viscoelasticity. Under the effect of positive inotrope, isoprenaline, cell-QCMsurface adhesion became stronger with increased Df and DR; CVI increased and the cells became stiffer. Whereas under the effect of negative inotrope, verapamil, cell-QCM surface adhesion became weaker with decreased Df and DR; CVI dropped and the cells became softer.Conclusion:QCMcan be used to monitor dynamic cell adhesion of different concentrations of H9C2 rat myocardial cells; and differentiate the effects of positive and negative inotropic drugs based on changes in cell adhesion strength and cells'' viscoelasticity, demonstrating its potential as a screening tool for cardiovascular drugs.