论文部分内容阅读
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 study the effect of self-made monoclonal antibody against intercellular adhesion molecule-1 (ICAM-1) on the in vitro and in vivo target of cardiomyocytes injured by biotin-avidin To bind, and observe its anti-inflammatory effect. Methods PFOB microspheres biotinylated, normal and conjugated anti-ICAM-1 monoclonal antibodies were prepared and their conjugation was detected by immunofluorescence technique. Primary rat cardiomyocytes were cultured in vitro and divided into TNF-α-treated group and untreated Group and fluorescence microscope were used to observe the direct and direct binding of biotinylated PFOB microspheres and target PFOB microspheres to TNF-α-stimulated cardiomyocytes, respectively. The model of myocardial ischemia-reperfusion in rats was established and divided into group A (myocardial ischemia-reperfusion rats + target microspheres), group B (myocardial ischemia-reperfusion rats + common microspheres), group C Mouse + targeted microspheres), group D (normal rats + normal microspheres), group E (myocardial ischemia-reperfusion rats + saline) and group F (sham operation). Fluorescence microscopy was used to observe the microstructure of the hearts frozen in group A-D. The serum levels of IL-8 in group A, B, E and F after 6h and 24h reperfusion were measured by liquid-phase microarray. RESULTS: ICAM-1 McAb was successfully coupled with PFOB microspheres with a coupling rate of 95%. A large number of green fluorescent microspheres were observed in the TNF-α-treated group in vitro, but only a few microspheres were observed in the untreated group ; In vitro direct targeting Whether or not cardiomyocytes were exposed to TNF-α, normal microspheres were not attached to the cardiac muscle cells, while a large number of targeted microspheres combined with myocardial cells under TNF-αinfection. Targeting and ordinary PFOB microspheres all played a certain anti-inflammatory effect on injured myocardium compared with saline group. Conclusion Anti-ICAM-1 targeting PFOB microspheres can directly and indirectly target the impaired cardiomyocytes that express ICAM-1 in vitro and in vivo and exert their anti-inflammatory effects.