Evaluation of the mechanism of apoptosis in cardiac myocyte with or without sympathetic innervation by Anthracycline

为阐明交感神经支配是否对蒽环类药物诱导的大鼠心肌细胞凋亡有保护作用，本实验对新生大鼠心肌细胞进行培养，并与交感神经节共培养。生长3天后，新生大鼠心肌细胞形成星状构型。交感神经节与心肌细胞共培养72小时后，可见明显的轴突。在每种培养中，单独的轴突与约10%的心肌细胞直接接触。用柔红霉素1μ摩尔或不加柔红霉素1 DNA摩尔处理培养的细胞。原位末端标记法和DNA梯带法检测到柔红霉素作用后8h出现细胞凋亡，12h达高峰。柔红霉素作用后，培养的心肌细胞和交感神经支配的培养心肌细胞的TUNEL阳性细胞数差异有统计学意义。但是，在培养的心肌细胞和交感神经支配的培养的心肌细胞中，只有很少的百分比的凋亡率被计算出来。接下来，我们研究了原癌基因bax和p53在培养的交感神经支配的心肌细胞和交感神经支配的心肌细胞中的表达。柔红霉素暴露后，培养细胞和共培养细胞在90℃时表达上调。此外，与交感神经支配的培养心肌细胞相比，柔红霉素作用于120℃培养的心肌细胞，直接引起DNA断裂的酶Caspase3的表达显著增加。因此，交感神经支配可通过保护蒽环类药物对心肌细胞的凋亡起保护作用。

To clarify whether sympathetic innervation protects the apoptosis by Anthracyclines in rat cardiac myocyte, we cultured 1 day old neonatal rat cardiac myocytes which was not innervated until 1 day old, and co-cultured cardiac myocytes with sympathetic ganglion. After 3 days of growth, neonatal rat ventricular myocytes developed a stellate configuration. When sympathetic ganglion were co-cultured with the myocytes for initial 72 hours in culture, axons were readily apparent. Individual axons made direct contact with about 10% of myocytes in each culture. These cultured cells were exposed with or without Daunorubicin 1 μ mol. Apoptosis were detected from 8 hours and peaked at 12 hours after Daunorubicin exposure by TUNEL method and DNA laddering. There were significant differences in the rare of TUNEL positive cells between cultured myocytes and sympathetic innervated cultured myocytes after Daunorubicin. But, only a little percent of apoptosis in cultured myocytes and sympathetic innervated cultured myocytes without Daunorubicin exposure were calculated.Next we evaluated the expression of Bax and p53 which were proto-oncogene and its transcriptional factor in cultured and sympathetic innervated myocytes with or without Daunorubicin. Both cultured and co-cultured cells equally up-regulated at 90 after Daunorubicin exposure. Moreover, we detected the expression of Caspase 3 which was enzyme for DNA fragmentation directly was significantly increased in cultured myocyte at 120 after Daunorubicin exposure as compared with those in sympathetic innervated cultured myocytes. Thus, sympathetic innervation could be protected apoptosis in cardiac myocyte by Anthracyclines.