【作者】 周卉芬；

【导师】 王伟；

【作者基本信息】 汕头大学 ， 心血管内科学， 2010， 硕士

【摘要】 目的:1.研究SIRTs(SIRT1-7)在大鼠心脏组织及H9c2心肌细胞的定位;2.初步探讨热量限制对心肌细胞SIRT1-7表达的影响。方法:3个月龄健康雄雌SD大鼠20只,随机分成两组:①正常对照组(ad libitum,AL)(n=10):随意进食;②热量限制组(Calorie Restriction,CR)(n=10):喂养量为对照组的60%,分别称体重。饲养3周后称体重,用质量分数2 %的戊巴比妥麻醉后处死,取出心脏,称重,沿心脏纵轴平均分为三份,心尖及心底部用于Western blot,对比两组SIRT1-7蛋白表达水平的变化;中间部分心脏用于免疫组织化学,定性检测SIRT1-7在大鼠心脏组织的定位。大鼠心肌细胞株H9c2在含葡萄糖4.5g/L的DMEM培养基中培养48h后传代随机分为两组:①正常对照组(Control,Con):继续在含葡萄糖4.5g/L的DMEM中培养;②热量限制组(CR):弃去旧培养液,加入含葡萄糖1g/L的DMEM。两组细胞在5% CO2、37℃条件下继续培养24h后终止。免疫细胞化学法定性检测SIRT1-7在H9c2的定位; RT-PCR法检测SIRT1-7 mRNA表达水平的变化。结果:1.实验前两组大鼠两两体重的差别无统计学意义。心脏质量指数(HMI)=心脏重量/大鼠体重。实验后CR组大鼠体重及HMI均显著低于AL组(p<0.05)。2.免疫组织化学法检测SIRT1-7在大鼠心肌细胞定位,结果表明:SIRT1在细胞核及细胞质均有大量表达,以细胞核表达为主;SIRT2、SIRT3、SIRT4、SIRT5主要在细胞质表达;SIRT6、SIRT7在细胞核及细胞质均有表达,主要在细胞核表达。3.免疫细胞化学法检测SIRT1-7在H9c2细胞表达定位,结果表明:SIRT1、SIRT2、SIRT7在细胞核及细胞质均有表达,以细胞核表达为主;SIRT3、SIRT4、SIRT5、SIRT6在细胞质表达为主。4. Western Blot法检测热量限制对大鼠心肌细胞SIRT1-7蛋白表达水平的影响,结果表明:CR组SIRT1,2,3,4,7蛋白表达水平与AL组比较显著增加(p<0.05);SIRT5,6的蛋白表达在两组中没有差异。5. RT-PCR法检测热量限制对H9c2细胞SIRT1-7 mRNA表达水平的影响,结果表明:其变化与大鼠心脏SIRT1-7蛋白表达水平的变化一致。SIRT1,2,3,4,7 mRNA表达水平与正常对照组比较明显增加(p<0.05),SIRT5,6 mRNA的表达在两组中没有差异。结论:1.在心肌细胞中,SIRT1在细胞核及细胞质均有大量表达,以细胞核表达为主;SIRT2,3,4,5主要在细胞质表达;SIRT6、SIRT7主要在细胞核表达。2.热量限制可同时激活心肌细胞SIRT1,2,3,4,7,而对SIRT5,6的表达没有影响;初步推测热量限制对心肌细胞的作用可能与SIRT1,2,3,4,7表达的上调有关。更多还原

【Abstract】 Objective:1. To investigate the cellular localization of SIRTs (SIRT1-7) both in rat cardiac tissues and H9c2 cells;2. To study the effects of calorie restriction on the expression of SIRTs in cardiomyocytes.Methods:Twenty 3-month-old SD rats of both sexes were randomized to either control group (n=10) or CR group (n=10), with five males and five females in each group. Rats in control group were fed ad libitum (AL), while rats in CR group were fed by 60% of AL. After 3 weeks, twenty rats were weighed and killed. Hearts were flushed with 0.9% normal saline solution before taken. The hearts were then sectioned into three parts, the middle part of the heart were used for immunohistochemistry, for detecting the cellular localization of SIRTs (SIRT1-7) in rats cardiac tissues. The same area of the heart from each rat including dual atriums and dual ventricles were used for immunohistochemistry. The rest parts of hearts were used for Western blot to investigate the protein levels of SIRTs.The H9c2 embryonic rat heart-derived cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100ml/L fetal bovine serum (FBS) and 4.5g/L glucose. The cells were trypsinized, plated (10 000-12 000cells/cm2) in culture dishes, then were randomly divided into two groups after culturing for 48 h, (1) normal control group(Con): without any treatment; (2) calorie restriction group (CR) : cells were incubated in DMEM containing 1g/L glucose. Both groups were incubated in 5% CO2 at 37℃for 24 h. Immunohistochemistry examination was used to determine the cellular localization of SIRTs (SIRT1-7) in H9c2 cells. The expressions of SIRT1-7 mRNA were detected by RT-PCR. Results:1. The differences in body weight between AL group and CR group was not significant before the study protocol. However, after maintenance on a CR diet for three weeks, the body weight (BW) and the heart mass index (HMI=Heart weight/BW) were significantly lower in the CR group when compared with the AL group (p<0.05).2. Each of the seven SIRT proteins was detected in cardiac tissues of SD rats both in AL group and CR group, evidenced by immunohistochemistry. SIRT1 is ubiquitous, as which was distributed in nucleus and cytoplasm. SIRT2, 3, 4, 5 were detected primarily in cytoplasm. SIRT6 and SIRT7 are mainly concentrated in the nucleus.3. A similar expression pattern of SIRT1-7 was observed from immunohistological study in H9c2 cells. Our results show that SIRT1, 3, 4, 5, 7 in H9c2 cells share the same distribution characteristics as in rat heart tissues. SIRT1 is localized both in nucleus and cytoplasm. SIRT3, 4, 5 were detected mainly in cytoplasm while SIRT7 was distributed in nucleus. Intriguingly, high density of SIRT2 in nucleus was observed while SIRT6 was detected primarily in cytoplasm.4. To explore the possible effects of CR on the expression of sirtuins in rat hearts, twenty SD 3-month-old rats were fed either AL or on a CR regimen for 3 weeks. We investigated whether protein levels of SIRT1-7 change after CR. Western blot analysis showed that CR caused SIRT1, 2, 3, 4, 7 statistically significant up-regulations (p<0.05), while the expression of SIRT5, 6 were unaffected.5. The expression pattern of SIRT1-7 mRNA consists with the results of protein, which further support the results mentioned above. H9c2 cells were cultured either in CR group (4.5g/L glucose) or in control group (1g/L glucose) for 24h separately. RT-PCR revealed that the expression of SIRT1, 2, 3, 4, 7 mRNA significantly increased in CR group compared with control group (p<0.05), but SIRT5, 6 were unaffected. Conclusion:1. SIRTs are expressed in the cardiomyocytes both in vivo and in vitro; SIRT1 is distributed in nucleus and cytoplasm, mainly in nucleus. SIRT2, 3, 4, 5 were detected primarily in cytoplasm. SIRT6 and SIRT7 are mainly concentrated in the nucleus.2. Calorie restriction has effects on cardiomyocytes by increasing the expression of SIRT1, 2, 3, 4, 7.更多还原