老龄大鼠心肌缺血预处理后αB-晶体蛋白表达变化的研究

Expression of alpha-B crystalline in myocardium after ischemia-preconditioning in old rats

目的:观察小分子热休克蛋白αB-晶体蛋白在青年大鼠及老年大鼠心肌缺血预处理后mRNA和蛋白表达的变化,探讨αB-晶体蛋白表达与老龄大鼠部分丧失缺血预处理保护作用的关系。方法:2～3月龄及24月龄SD大鼠各24只,雌雄不限,分别按照随机分组原则分为老年对照组(n=4)、老年预处理组(n=20)、青年对照组(n=4)、青年预处理组(n=20)。两预处理组予停灌5min、复灌5min,连续3次,制备缺血预处理模型,于末次灌注后0、5、15、45和60 min,两对照组于假手术后持续灌流60 min取左心室缺血的前壁与非缺血的后壁心肌组织分别匀浆,采用RT-PCR方法观察大鼠αB-晶体蛋白mRNA在不同时间点的表达情况;离心取上清及沉淀蛋白,用Western Blot方法检测缺血预处理不同时间αB-晶体蛋白在可溶性蛋白及不溶性细胞骨架中含量的改变;新鲜冰冻心肌组织采用免疫荧光方法标记αB-晶体蛋白,在激光共聚焦显微镜下观察αB-晶体蛋白的分布情况。结果:①老龄大鼠αB-晶体蛋白mRNA的基础表达量与青年大鼠相比未见明显差异;缺血预处理后约15min,其表达在老龄大鼠及青年大鼠均有所增加;②青年大鼠心脏缺血预处理后即刻前壁缺血组织αB-晶体蛋白从胞浆(上清液)移至不溶性成分细胞骨架(沉淀),至15 min时大部分移到沉淀中,在45 min时则几乎全部复位到上清中,而老龄大鼠心肌中αB-晶体蛋白在各时间点均未发生明显移位。③免疫荧光检查显示青年大鼠αB-晶体蛋白在缺血预处理后15 min向Z线及润盘部位聚集;老年大鼠中αB-晶体蛋白移位程度与成年大鼠相比明显减弱。结论:老年大鼠心肌组织中αB-晶体蛋白移位能力降低,从而减弱了与各相应蛋白结合的能力,这可能是老年大鼠缺血预处理保护作用减弱的重要原因之一。

Objective： To observe the changes in the expression of alpha-B crystalline, a kind of small-molecular heat shock protein, in the myocardium after ischemia-preconditioning （I/P） in aged and young rats, and investigate the relationship between alpha-B crystalline expression and partial loss of I/P protection against ischemia- reperfusion damage. Methods.. Forty-eight SD rats,among them 24 were 2-3 months old and 24 were 24 months old, were randomly divided into four groups： aged control group （n=4）, young control group （n=4）, aged model group （n= 20） and young model group （n= 20）. I/P model was replicated in aged and young model groups, in which rats received 5-minute ischemia and 5-minute reperfusion for 3 times. The two control groups did not undergo ischemia. Heart tissues were collected at 0,5,15,45,60 minutes after I/P-induced damage. RT-PCR and Western blot analysis were used to examine the expression of alpha-B crystalline mRNA and protein, respectively. Double immunofluorescence labeling-confocal microscopy was used to observe the translocation of alpha-B crystalline after I/P. Results：（1）Results showed that the basic expression of alpha-B crystalline mRNA in aged was not significantly different from that of young rats. Whereas the expression of alpha-B crystalline mRNA increased after ischemia for 15 minutes both in young and aged rats. （2）The alpha-B crystalline protein in anterior wall of left ventricle was transferred from the cytosol to sites of the myofibrillar system immediately after I/P in young rats, but not in aged rats,and almost all of it returned to the supernatant after treating of ischemia for about 15 minutes,and it almost transferred to cytosol after about 45 minutes. （3）Immunohistology indicated that after ischemia pretreatment for 15 minutes, alpha-B crystalline protein was shifted to Z line and I band, while the shift level of alpha-B crystalline protein was significantly weak in aged rats. Conclusion： The present results demonstrate that translocation ability of alpha-B crystalline in aged rat is reduced, which may explain why the heart of aged rats partly lose the protective effect of I/P against ischemic injury of the myocardium.