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目的设计一套用于心肌组织工程的双相脉冲电刺激(BPES)系统,并研究BPES对体外培养的脂肪干细胞(ADSC)向心肌细胞样细胞分化的影响。方法分离和培养SD大鼠ADSC,通过流式细胞术对细胞表型进行鉴定,通过成脂、成骨分化证明其多向分化潜能。选择第3代细胞随机分为2组:BPES组接种24 h后,给予BPES(脉宽2 ms、幅度2 V、频率2 Hz、刺激时间2 h/d,连续刺激3、7、14 d),每3 d换1次液;对照组培养条件与BPES组相同,但不给予BPES。通过倒置相差显微镜观察细胞形态变化,MTT法检测细胞增殖,免疫荧光技术检测细胞同源盒蛋白Nkx-2.5和连接子蛋白43(CX43)的水平。结果流式细胞术证明分离的细胞是ADSC,成脂诱导后并用油红O染色,可见细胞内脂滴被染成红色,成骨诱导后碱性磷酸酶(ALP)和Von Kossa染色,可见钙结节特征;细胞增殖无明显变化,BPES组Nkx-2.5和CX43蛋白表达量明显高于对照组。结论成功设计了可用于组织工程的BPES系统,并证明BPES可在体外促进ADSC向心肌细胞样细胞分化。
Objective To design a biphasic pulsed electrical stimulation (BPES) system for myocardial tissue engineering and to study the effects of BPES on cardiomyocyte-like cell differentiation of adipose-derived stem cells (ADSCs) cultured in vitro. Methods The ADSCs of SD rats were isolated and cultured. The cell phenotypes were identified by flow cytometry. The differentiation potential of ADSCs was proved by adipogenic and osteogenic differentiation. The third generation cells were randomly divided into two groups: BPES group was given BPES (pulse width 2 ms, amplitude 2 V, frequency 2 Hz, stimulation time 2 h / d, continuous stimulation 3, 7 and 14 d) , Once every 3 d for liquid; control group culture conditions and BPES group same, but not give BPES. Cell morphological changes were observed by inverted phase contrast microscope. Cell proliferation was detected by MTT assay. Cell homeobox proteins Nkx-2.5 and CX43 were detected by immunofluorescence staining. Results Flow cytometry showed that the isolated cells were ADSCs. After adipogenic induction and staining with Oil Red O, the lipid droplets in the cells were stained red. After osteogenic induction, ALP and Von Kossa staining showed that calcium Nodules characteristics; no significant changes in cell proliferation, BPES group Nkx-2.5 and CX43 protein expression was significantly higher than the control group. Conclusion The BPES system which can be used in tissue engineering has been successfully designed and proved that BPES can promote the differentiation of ADSC into cardiomyocyte-like cells in vitro.