培养成年大鼠心肌细胞存活的形态标志

目的：通过探寻增加培养成年大鼠心肌细胞存活率以及防止再分化的方法,揭示培养成年大鼠心肌细胞存活的形态标志。方法：采用Langendorff系统灌流心脏,胶原酶消化法分离成年大鼠心肌细胞,分3组进行细胞培养：①基础培养液＋凋亡抑制剂;②基础培养液＋5%胎牛血清;③基础培养液＋5%胎牛血清＋凋亡抑制剂。结果：①培养前3天杆状心肌细胞比例逐渐降低,无血清培养组比血清培养组降低程度大。培养前3天凋亡率逐渐升高,无血清培养组比血清培养组凋亡率高,加入凋亡抑制剂对凋亡率无影响。②有血清培养2~3天的成年大鼠心肌细胞闰盘部位伸出伪足,促使细胞贴壁生长;当培养至第6天时,细胞侧面也伸展出贴壁的伪足,细胞丧失杆状形态,横纹消失。而无血清培养的细胞无伪足生成,随着培养时间增加,细胞末端变圆钝,横纹变模糊。凋亡抑制剂对伪足形成率无影响。③培养存活的成年大鼠心肌细胞骨架重排,发生再分化。④血清培养组细胞胞内核间距离随着培养时间的增加而减小,无血清培养组则保持不变。结论：成年大鼠心肌细胞培养至第2~3天时,闰盘部位形成伪足是细胞存活的形态标志,加入血清是伪足形成的必要条件。

Morphological signs of survival cultured adult rat cardiomyocytes

Objective： To clarify the key morphological signs for the survival of adult rat cardiomyocytes in primary culture.Methods： The adult rat hearts were retrogradely superfused by Langendorff apparatus.Cardiomyocytes were digested by collagenase I and cultured in three groups： ①Serum free medium ＋ BA（Bongkrekic acid,apoptotic inhibitor）,②5% serum medium,and ③5% serum medium ＋ BA.The morphological alterations were observed and the percentage of rod-shaped cardiomyocytes,the apoptotic rate of cells,the rate of pseudopodium formation and the nuclear distances of cardiomyocytes were detected during culture.Results： ①The percentage of rod-shaped cardiomyocytes decreased gradually in the first 3 days of cell culture.The percentage of rod-shaped cardiomyocytes cultured without fetal bovine serum（FBS） decreased more rapidly than those cultured with FBS.No differences were noticed between with and without the addition of apoptotic inhibitor BA.The apoptotic rate of cardiomyocytes increased in the first 3 days of cell culture,and the apoptotic rate of cells cultured without FBS increased more than that cultured with FBS.Also BA had no effect on apoptotic rate.②Cardiomyocytes cultured with FBS spread from the intercalated disk and extended pseudopodium on the second or third day of cell culture.Cardiomyocytes with thin membranous pseudopodium deve-loped would survive and spread laterally at the 6th day of culture.Cells with the elongated morphology gradually spread extensively and took on a spheroidal shape.Myofibrils gradually lost their parallel.Cells cultured without FBS had no pseudopodium formation.The intercalated disk of cells gradually changed blunt.There was no effect on the rate of pseudopodium formation when added with apoptotic inhibitor BA.③Cytoskeletal remodeling occurred in survived cardiomyocytes.After 6 days of culture,cardiomyocytes exhibited characteristic of redifferentiation.④The distance between nuclei decreased in a single cardiomyocyte cultured with FBS for the cytoskeletal reconstruction,whereas it remained unchanged in cardiomyocytes cultured without FBS.Conclusion： We clarify the pseudopodium developed on the second or third day of cell culture will be the critical morphological signs of survival cultured adult rat cardiomyocytes.It is necessary to add FBS for the formation of pseudopodium.