原子力显微镜单分子力谱研究ICAM-1/CD11b相互作用及药物干预的影响

ICAM-1/CD11b interactions and effect of drug intervention by AFM single molecule force spectroscopy

目的利用绿色荧光蛋白(GFP)标记人细胞间黏附分子-1(ICAM-1),观察GFP是否影响ICAM-1在细胞膜表面的定位,实现ICAM-1可视化。应用原子力显微镜(AFM)单分子力谱研究ICAM-1/CD11b相互作用,并探讨瑞舒伐他汀(RSV)及单抗类药物干预的影响。方法从人脐静脉内皮细胞(HUVEC)中提取总RNA,设计合成PCR引物(两端分别含有Bam HⅠ、HindⅢ特异性酶切位点),使用RT-PCR扩增获取ICAM-1cDNA编码区序列,经测序分析筛选正确序列,与pEGFP-N1表达载体相接,即得到基于GFP的重组质粒pICAM-1-GFP,脂质体介导转染COS-7细胞,通过荧光显微镜观察融合蛋白pICAM-1-GFP在COS-7上的定位。AFM单分子力谱测量该细胞模型上单对黏附分子ICAM-1/CD11b的相互作用力值,应用RSV及抗ICAM-1单克隆抗体干预,检测黏附力值是否有变化。结果序列分析RT-PCR扩增获得的片段为人ICAM-1的cDNA编码区,pICAM-1-GFP发出绿色荧光定位于COS-7细胞表面。单对黏附分子ICAM-1/CD11b的相互作用力值约为42pN,RSV干预对此值无明显影响,而抗ICAM-1单克隆抗体则有效降低了此单对黏附分子亲和力。结论荧光蛋白GFP能融合于ICAM-1的C端,且不影响ICAM-1在COS-7细胞膜表面的定位表达。RSV不能通过直接阻断单分子ICAM-1或CD11b影响其相互作用,可能是通过有效抑制ICAM-1表达量的增加从而发挥抗动脉粥样硬化炎症进程作用。抗ICAM-1单克隆抗体有效降低黏附分子亲和力,提示单抗类药物在抗炎治疗领域的应用前景。该研究建立的方法模型可作为活细胞体系探讨单对黏附分子亲和力及药物干预影响的重要研究手段。

Objective To observe whether green fluorescent protein(GFP)affects the localization of human intercellular cell adhesion molecule-1(ICAM-1)on the cell membrane surface,visualize ICAM-1 study the interactions of ICAM-1/CD11b and to explore the effects of rosuvastatin and monoclonal antibody intervention. Methods Total RNA was extractedfrom human umbilical vein endothelial cells(HUVECs),and PCR primers were designed and synthesized(Bam HⅠ andHind Ⅲ specific restriction sites were at both ends). The sequence of ICAM-1 cDNA coding region was amplified by RT-PCR,and the correct sequence was screened by sequencing analysis. The recombinant plasmid pICAM-1-GFP wasobtained by ligation with pEGFP-N1 expression vector,and liposome-mediated transfection was performed. The locationof the fusion protein pICAM-1-GFP on COS-7 was observed by fluorescence microscopy.The atomic force microscope(AFM)single molecule force measurement was used to measure the interaction force of the single-pair adhesion moleculeICAM-1/CD11b in the COS-7 cell model that was intervened in with rosuvastatin(RSV)and anti-ICAM-1 monoclonalantibody. Results The fragment obtained by RT-PCR amplification of sequence analysis was the cDNA coding region of human ICAM-1. The recombinant plasmid pICAM-1-GFP fusion protein emitted green fluorescence and was localizedon the surface of COS-7 cells.The interaction force of the single-pair adhesion molecule ICAM-1/CD11b was about 42 pN,and the RSV intervention had no significant effect on this value,while the anti-ICAM-1 monoclonal antibody effectivelyreduced the affinity of the single pair of adhesion molecules. Conclusion Fluorescent protein GFP is able to fuse tothe C-terminus of ICAM-1,but does not affect the localized expression of ICAM-1 on the surface of COS-7 cell membrane.RSV cannot affect its interaction by directly blocking single molecule ICAM-1 or CD11b,and may play an anti-atheroscleroticrole in the inflammatory process by effectively inhibiting the increase in ICAM-1 expression. The anti-ICAM-1 monoclonalantibody effectively reduces the affinity of the molecule,suggesting the prospect of application of the monoclonal antibodyin the field of anti-inflammatory therapy. The method model established in this study can be used as an important means forthe study of the affinity of single-adhesion molecules and drug interventions as a living cell system.