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

为了阐明交感神经支配是否保护蒽环类药物对大鼠心肌细胞的凋亡作用，我们培养了1日龄的未受神经支配的新生大鼠心肌细胞，并将心肌细胞与交感神经节共培养。生长3天后，新生大鼠心室肌细胞形成星状结构。交感神经节与肌细胞共培养72小时，轴突明显。在每种培养中，单个轴突与约10%的肌细胞直接接触。采用TUNEL法和DNA阶梯法，分别用柔红霉素（1 μ mol）和不加柔红霉素（1 μ mol）对培养细胞进行处理，8 h开始检测细胞凋亡，12 h达到凋亡高峰。柔红霉素作用后，培养肌细胞与交感神经培养肌细胞的TUNEL阳性细胞数量差异有统计学意义。但是，在没有柔红霉素暴露的情况下，仅计算培养肌细胞和交感神经细胞的凋亡百分比。接下来，我们评估了原癌基因Bax和p53及其转录因子在加或不加柔红霉素的培养和交感神经支配的肌细胞中的表达。在柔红霉素暴露后，培养细胞和共培养细胞均在90时上调。此外，我们检测到，与交感神经支配的培养肌细胞相比，暴露于柔红霉素120时，培养肌细胞中Caspase 3的表达显著增加，Caspase 3是DNA直接断裂的酶。由此可见，蒽环类药物可保护交感神经的凋亡。

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.