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诱导的大鼠心肌细胞凋亡，我们培养了1日龄新生大鼠心肌细胞，这些心肌细胞直到1日龄才被支配，并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构型。当交感神经节与肌细胞共培养最初72小时时，轴突很容易出现。在每种培养物中，单个轴突与约10%的肌细胞直接接触。这些培养的细胞暴露于柔红霉素1 μ mol或不暴露于柔红霉素1 μ mol。TUNEL法和DNA梯状条带法检测到柔红霉素作用后8 h开始出现凋亡，12 h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的心肌细胞中TUNEL阳性细胞的稀少程度有显著性差异。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，没有柔红霉素暴露，只有一小部分细胞凋亡的计算。接下来，我们评估了Bax和p53的表达，这是原癌基因及其转录因子在培养的和交感神经支配的心肌细胞与柔红霉素或无。在柔红霉素暴露后，培养的和共培养的细胞在90时同样上调。另外，我们还检测到，与交感神经支配的培养心肌细胞相比，柔红霉素暴露后120 ℃培养的心肌细胞中直接进行DNA片段化的酶Caspase 3的表达显著增加。蒽环类药物对交感神经支配的心肌细胞凋亡有保护作用。

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.