衣霉素诱导乳鼠原代心肌细胞凋亡的实验研究

目的探讨衣霉素在诱导大鼠心肌细胞凋亡中的作用和可能的凋亡通路。方法采用消化贴壁法分离乳鼠原代心肌细胞,应用衣霉素对乳鼠原代心肌细胞进行凋亡诱导,通过MTT、DNA梯带、Hoechst染色等方法对心肌细胞凋亡进行定性和定量检测,选出合适的诱导浓度和诱导时间。用Western印迹法检测凋亡过程中Grp78、caspase-12蛋白酶的表达变化。结果衣霉素5μg/mL诱导24h后可引起30%心肌细胞凋亡,并出现典型的凋亡形态改变;在TM（5μg/mL）诱导3h和24h时分别有Grp78和caspase-12蛋白表达。TM诱导48h表现出DNA弥散样变化。结论衣霉素可通过内质网应激通路诱导心肌细胞凋亡,5μg/mL诱导24h可作为衣霉素建立乳鼠原代心肌细胞内质网应激凋亡模型的合适条件。

Apoptosis of primary neonatal rat cardiac muscle cells induced by tunicamyocin

Objective To investigate the role of tunicamyocin（TM） in inducing apoptosis of cardiac muscle cells in rats and the possible apoptosis pathway.Methods Primary cardiac muscle cells were isolated from neonatal rats with the modified 2-step collagenase digest method.Apoptosis of these cells, induced by TM at an appropriate concentration and incubated for a certain time, was detected by MTT assay, DNA ladder and Hoechst staining.Expression of Grp78 and procaspase-12 in the process of apoptosis was detected by Western blot.Results Apoptosis with typical morphologic changes occurred in 30% of cardiac muscle cells 24 hours after induction with TM at the concentration of 5μg/mL.The expression of Grp78 and caspase-12 and diffuse changes in DNA smear could be detected 3, 24, and 48h, respectively, after induction with TM.Conclusion Apoptosis of primary cardiac muscle cells in rats can be induced by TM through the endoplasmic reticulum stress-associated pathway, 5μg/mL TM and 24-hour incubation may be the proper conditions for establishing an apoptosis model.