CAJ | 학술논문

目的建立简单实用和重复性好的新生小鼠心肌细胞体外培养方法,构建体外心肌细胞模型用于外源化合物的心肌毒性评价.方法取当天出生的小鼠心脏,用胰蛋白酶、Ⅱ型胶原酶和分散酶消化心室肌组织,用差速贴壁和化学方法纯化心肌细胞,用此细胞模型评价镰刀菌毒素丁烯酸内酯(BUT)的心肌毒性作用.结果新生小鼠心肌细胞培养3 d后,大部分细胞开始搏动,随培养时间的延长,心肌细胞胞体逐渐增大.经α-actin抗体免疫组织化学鉴定,心肌细胞的纯度为96%.持续培养90 d后,心肌细胞仍保持较好的搏动频率.经BUT染毒后,心肌细胞存活率随BUT浓度的增加而逐渐下降,对心肌细胞的损伤在形态上主要表现为细胞水肿、空泡样变性、肌纤维断裂等.结论该实验建立的新生小鼠心肌细胞体外培养方法,单细胞收获率和心肌细胞纯度高,心肌细胞搏动时间长.BUT对心肌细胞有较强的毒性作用并呈明显的量-效关系.
목적 건립간단실용화중복성호적신생소서심기세포체외배양방법,구건체외심기세포모형용우외원화합물적심기독성평개.방법 취당천출생적소서심장,용이단백매、Ⅱ형효원매화분산매소화심실기조직,용차속첩벽화화학방법순화심기세포,용차세포모형평개렴도균독소정희산내지(BUT)적심기독성작용.결과 신생소서심기세포배양3 d후,대부분세포개시박동,수배양시간적연장,심기세포포체축점증대.경α-actin항체면역조직화학감정,심기세포적순도위96%.지속배양90 d후,심기세포잉보지교호적박동빈솔.경BUT염독후,심기세포존활솔수BUT농도적증가이축점하강,대심기세포적손상재형태상주요표현위세포수종、공포양변성、기섬유단렬등.결론 해실험건립적신생소서심기세포체외배양방법,단세포수획솔화심기세포순도고,심기세포박동시간장.BUT대심기세포유교강적독성작용병정명현적량-효관계.
Objective To establish a method for culturing neonatal mice cardiomyocytes, and construct an in vitro model of cardiomyocytes for assessing the cardiac toxicity of chemicals. Methods Hearts of neonatal mice of 1 day old were digested with enzyme mixture of trypsin/collagenase type Ⅱ/dispase and the cell suspensions were pre-plated to flask for a short time and then seeded on coated dishes. The cultured cells were treated with 5-fiuorine-deoxy-uridine(15 μg/ml)and uridine(35 μg/ml)to enrich cardiomyocytes that were identified according to the morphology and immunocytochemistry of α-actin antigen. Cardiac myocytes were incubated with 0-64 μg/ml of a fusarium mycotoxin butenolide(BUT) for 12 h. Cell viability was then evaluated by MTT assay. Microscopic observation showed that BUT induced significant morphological changes including cellular swelling, vacuolation and breakage of muscle fibers. Results It was found that 96% of the cultured cells were cardiomyocytes and the myocytes kept beating after 90 days of culture. Concentration-dependent decreases in cell viability following exposure of cardiac myocytes to BUT were observed. Conclusion The results indicated that relatively pure primary culture of neonatal mice cardiomyocytes is successfully established. BUT possesses the potential to induce myocardial toxicity.