Hypoxia reoxygenation induces premature senescence in neonatal SD rat cardiomyocytes
Hypoxia reoxygenation induces premature senescence in neonatal SD rat cardiomyocytes
复制标题
缺氧复氧诱导新生SD大鼠心肌细胞早衰
DOI：
10.1111/j.1745-7254.2007.00488.x
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发表时间：
2007-01-01
影响因子：
8.2
通讯作者：
Cao, Ke-jiang
中科院分区：
文献类型：
--
作者：
Zhang, Feng-xiang;Chen, Ming-long;Cao, Ke-jiang
摘要：探讨缺氧复氧是否会诱导新生儿SD大鼠心肌细胞过早衰老。方法：从新生SD大鼠心脏分离心肌细胞，采用免疫组织化学方法进行鉴定。对照培养物在湿度为5% CO2和95%空气的37°C环境中培养。将缺氧培养物在充满1% O2、5% CO2和平衡N2的模块化培养箱中孵育6小时。再氧培养物分别在1% O2和5% CO2中孵育6小时，然后在21%氧气中孵育4、8、12、24和48小时。用溴脱氧尿苷标记法测定细胞增殖。电镜下观察心肌细胞超微结构。采用衰老β-半乳糖苷酶染色试剂盒测定β-半乳糖苷酶活性。实时定量PCR检测p16INK4a和端粒酶逆转录酶（TERT） mrna水平。免疫组化法检测TERT蛋白表达。端粒酶活性检测采用Telo TAGGG端粒酶PCR elisa +试剂盒。结果：初始培养为免疫组织化学鉴定的纯心肌细胞。缺氧复氧组BrdU阳性细胞比例显著降低（P<0.01）。缺氧复氧条件下，线粒体出现脱水；p16INK4a和TERT mRNA水平、β-半乳糖苷酶活性、TERT蛋白表达和端粒酶活性均显著或极显著升高（P<0.01或P<0.05）。结论：缺氧复氧可诱导新生SD大鼠心肌细胞过早衰老。TERT虽显著升高，但不能阻断衰老。
AbstractAim:To investigate whether hypoxia reoxygenation induces premature senescence in neonatal Sprague-Dawley (SD) rat cardiomyocytes.Methods:Cardiomyocytes were isolated from neonatal SD rat heart and identified by immunohistochemistry. The control cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The hypoxic cultures were incubated in a modular incubator chamber filled with 1% O2, 5% CO2, and balance N2 for 6 h. The reoxygenated cultures were subjected to 1% O2 and 5% CO2 for 6 h, then 21% oxygen for 4, 8, 12, 24, and 48 h, respectively. Cell proliferation was determined using bromodeoxyuridine labeling. The ultrastructure of cardiomyocytes was observed by using an electron microscope. β-Galactosidase activity was determined by using a senescence β-galactosidase Staining Kit. p16INK4a and telomerase reverse transcriptase (TERT) mRN A levels were measured by real time quantitative PCR. TERT protein expression was determined by immunohistochemistry. Telomerase activities were assayed by using the Telo TAGGG Telomerase PCR ELISAplus kit.Results:The initial cultures consisted of pure cardiomyocytes identified by immunohistochemistry. The proportion of BrdU positive cells was reduced significantly in the hypoxia reoxygenation-treated group (P<0.01). Under the condition of hypoxia reoxygenation, mitochondrial dehydration appeared; p16INK4a and TERT mRNA levels, β-galactosidase activity, TERT protein expression and telomerase activities were all significantly increased (P<0.01 or P<0.05).Conclusion:These data indicate that premature senescence could be induced in neonatal SD rat cardiomyocytes exposed to hypoxia reoxygenation. Although TERT significantly increased, it could not block senescence.