p21 WAF1 and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes.
p21 WAF1 and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes.
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p21 WAF1 和缺氧/复氧诱导的 H9c2 心肌细胞过早衰老。
DOI：
10.5603/fhc.2011.0063
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发表时间：
2011-10
期刊：
影响因子：
--
通讯作者：
Chen, Ming-Long
中科院分区：
文献类型：
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作者：
Wang, Dan;Zhang, Yu-Zhen;Yang, Bing;Zhang, Feng-Xiang;Cao, Ming-Yong;Wang, Cheng;Chen, Ming-Long
我们以前曾报道过缺氧/复氧诱导的新生大鼠心肌细胞早衰。在本研究中，我们研究了p21（WAF 1）（p21）在缺氧/复氧诱导的衰老中的作用，使用H9 c2细胞。构建过表达野生型p21（WAF 1）的质粒和表达靶向p21的小发夹RNA（shRNA）的质粒（WAF 1），并转染H9 c2细胞以控制p21的表达。缺氧/复氧条件为1%O2和5%CO（2），用N（2）平衡培养箱6 h（缺氧6 h），然后21%氧气8 h（复氧8 h）。流式细胞仪检测细胞周期。使用β-半乳糖苷酶染色评估衰老。Western blotting检测p53、p21、p16（INK 4a）和cyclin D1的表达。缺氧6 h，p21过表达细胞G1期分布明显扩大，β-半乳糖苷酶活性增强，cyclin D1表达降低，而p21敲除细胞G1期分布明显扩大，p53表达增强。复氧8h时，p21沉默组G1期细胞比例减少，β-半乳糖苷酶活性减弱，16（INK 4a）表达降低，cyclin D1表达升高，而过表达组无明显差异。总之，这些数据表明，p21（WAF 1）是重要的缺氧阶段，但不是复氧阶段，在H9 c2衰老过程中。
We have previously reported on hypoxia/reoxygenation-induced premature senescence in neonatal rat cardiomyocytes. In this research, we investigated the effects of p21(WAF1) (p21) in hypoxia/reoxygenation-induced senescence, using H9c2 cells. A plasmid overexpressing wild type p21(WAF1) and a plasmid expressing small hairpin RNA (shRNA) targeting p21(WAF1) were constructed, and transfected into H9c2 cells to control the p21 expression. Hypoxia/reoxygenation conditions were 1% O2 and 5% CO(2), balancing the incubator chamber with N(2) for 6 h (hypoxia 6 h), then 21% oxygen for 8 h (reoxygenation 8 h). Cell cycle was examined using flow cytometry. Senescence was assessed using β-galactosidase staining. The expression of p53, p21, p16(INK4a), and cyclin D1 was assayed using Western blotting. At hypoxia 6 h, cells overexpressing p21 had a larger G1 distribution, stronger β-galactosidase activity, and lower cyclin D1 expression compared to control cells, while the opposite results and higher p53 expression were obtained in p21-knockdown cells. At reoxygenation 8 h, p21-silenced cells had a smaller percentage of G1 cells, weaker β-galactosidase activity and lower 16(INK4a) expression, and higher cyclin D1 expression, but the overexpression group showed no difference. Taken together, this data implies that p21(WAF1) is important for the hypoxia phase, but not the reoxygenation phase, in the H9c2 senescence process.