抗ICAM-1液态氟碳微球靶向结合损伤心肌细胞的体内外实验及其抗炎作用研究

Study of the targeted combination and anti-injury effect of anti-ICAM-1 targeted PFOB particles in injured myocardial cells

目的采用自制的靶向液态氟碳(PFOB)微球联合细胞间粘附分子-1(ICAM-1)单克隆抗体通过生物素-亲和素作用实现与大鼠损伤心肌细胞的体外及体内靶向结合,并观察其抗炎效果。方法制备生物素化、普通及耦联抗ICAM-1单克隆抗体的PFOB微球,免疫荧光技术检测其偶联情况;体外培养大鼠原代心肌细胞,分为TNF-α处理组和非处理组,荧光显微镜分别观察生物素化的PFOB微球及靶向PFOB微球对TNF-α刺激后心肌细胞的间接、直接靶向结合。建立大鼠心肌缺血再灌注模型,分为A组(心肌缺血再灌注大鼠+靶向微球)、B组(心肌缺血再灌注大鼠+普通微球)、C组(正常大鼠+靶向微球)、D组(正常大鼠+普通微球)、E组(心肌缺血再灌注大鼠+生理盐水)、F组(假手术)。荧光显微镜观察A～D组心脏冰冻切片微球结合情况;液相芯片技术检测A、B、E、F组再灌注6h及24h血清中IL-8含量。结果 ICAM-1单抗与PFOB微球成功耦联,耦联率达95%;体外间接靶向TNF-α处理组可见大量绿色荧光微球结合于心肌细胞周围,而非处理组仅见少许微球;体外直接靶向无论心肌细胞是否暴露于TNF-α,普通微球均未附着在心肌细胞周围,而大量靶向微球与TNF-α损伤下的心肌细胞相结合。体内直接靶向A组心肌细胞见微球结合,而B、C、D组则无;相对于生理盐水组,靶向和普通PFOB微球都对受损心肌起到了一定的抗炎作用。结论抗ICAM-1靶向PFOB微球可间接、直接靶向结合体外及体内高表达ICAM-1的受损心肌细胞,并可发挥其抗炎作用。

Objective To investigate the vitro and vivo targeted combination and anti-inflammatory effects of home-made anti-ICAM-1 targeted PFOB particles in rat model with myocardial reperfusion injury . Methods With the biotinylated, normal and anti-ICAM-1 targeted PFOB particles, the combination of ICAM-1 antibody with PFOB particles was evaluated by immunofluoreseent assay;Neonatal rat cardiomyocytes were cultured in vitro and divided into TNF-α activated group and non-activated group, The indirect and direct targeted combination was tested by immunofluorescence assay. SD rat models with myocardial ischemia-reperfusion injury were built up and allocated into 6 groups; Group A （reperfusion model with targeted PFOB particles）, Group B （reperfusion model with untargeted PFOB）, Group C （normal control with targeted PFOB particles）, Group D （normal control with untargeted PFOB）, Group E （reperfusion model with normal saline）, Group F （sham operation group）. The targeted combination was tested by immunofluorescence assay. The content of IL-8 was tested after the 6h and 24h of reperfusion by liquid chip technology in group A, B,E, and F. Results ICAM-1 antibody was successfully coupled with PFOB particles with coupling rate around 95% In vitro indirect part, there were amount of biotinylated PFOB particles in TNF-α activated group, while few particles in non-activated group; In vitro direct part, no matter whether the myocardial cells exposed to TNF-α, normal PFOB particles were not adhered to the myocardial cells, while a large number of targeted PFOB particles combined with the TNF-α activated cells. In vivo direct part, targeted combination was observed in group A, the targeted and untargeted PFOB particles both showed the anti-inflammation effects compared with the normal saline group. Conclusion The indirect and direct specific combination of anti-ICAM-1 targeted PFOB particles with injured myocardium as well as its anti-inflamma- tion effects were observed in our study.