微管蛋白在体外牵张刺激诱导心肌细胞肥大反应中的作用

Role of tubulin in hypertrophic response of cardiac myocytes induced by stretch in vitro

目的 探讨细胞骨架微管蛋白在牵张刺激心肌细胞肥大反应中的作用。方法 通过体外牵张培养的心肌细胞 ,采用3H 亮氨酸掺入法和四甲基偶氮唑盐微量酶反应比色法反映心肌细胞肥大指标和活心肌细胞数目 ,激光共聚焦定性、定量微管蛋白的合成和分布情况。结果 2 0 %的持续性牵张引起心肌细胞的3H 亮氨酸掺入量 [12h为 (1870 0± 112 5 )cpm ,2 4h为 (2 0 15 5± 193 7)cpm],与对照组 [12h为 (112 2 7± 12 6 5 )cpm ,2 4h为 (12 10 7± 2 2 2 7)cpm]比较显著增加 ,微管解聚剂秋水仙素 (4μmol L)可以明显抑制3H 亮氨酸掺入量 [12h为 (12 92 7± 14 2 6 )cpm ,2 4h为 (132 7 8± 97 4 )cpm]。牵张 12h即可见微管蛋白荧光强度增强 ,2 4h部分细胞呈现微管蛋白结构坍塌 ,纹理模糊 ,荧光强度分布不均 ,微管蛋白最高光密度值 16 0 0 (任意单位 ) ,可被 4 μmol L秋水仙素抑制。 结论 细胞骨架微管蛋白系统是牵张刺激诱导心肌细胞肥大反应的细胞内信号传递途径之一。

Objective To study the role of tubulin in the stretch-induced hypertrophic response of cardiac myocytes in vitro. Methods On the stretch model of cultured cardiac myocytes, MTT methods and 3H-leucine incorporation were used to calculate the survived cells number and measure the protein synthesis of cardiac myocytes. Furthermore, the confocal laser scanning microscopy was used to qualify and quantify the pixel intensity and distribution of the tubulin image. Results The radioactivity of 3H-leucine incorporated in the stretch-stimulated cardiac myocytes [(1870.0±112.5) cpm for 12 h and (2015.5±193.7) cpm for 24 h, P<0.05] was significantly higher than that in control[(1122.7±126.5)cpm for 12 h and (1210.7±222.7)cpm for 24 h] stimulated, but this increase could be restrain by 4 μmol/L colchicum. The pixel intensity and distribution of the tubulin image was increased for 12 h stretch and the OD value further to 1600(arbitrary unit). The changes were partially recovered by 4 μmol/L colchicum. Conclusion The tubulin way play a role in the stretch-induced hypertrophic response and play an intermediate in the stretch-stimulated expression of c-myc mRNA and protein in cardiac myocytes.