SIRT1 基因在衰老雌鼠卵巢中的表达变化 *

Expressional alteration of SIRT1 in ovarian of aging rats *

摘要:

目的??探讨 SIRT1 基因在衰老雌鼠卵巢中的表达变化。方法??选用 30 只清洁级 SD 雌性大鼠为研究 对象，分别于 6、12、24、48 及 72 周股动脉取血处死。每 3 天对雌鼠进行称重并记录，采用二硫代二硝基苯甲 酸法检测谷胱甘肽过氧化物酶（GSH-Px）活性，采用 Fe 3+ 还原法检测总抗氧化能力（T-AOC） ，采用酶联免疫 吸附试验（ELISA）检测血清中促黄体激素（LH） 、促卵泡激素（FSH） 、雌二醇（E 2 ）水平，光学显微镜观察 卵巢组织结构有无形态学改变，采用实时荧光定量聚合酶链反应（qRT-PCR）检测 SIRT1?mRNA，免疫组织 化学法检测SIRT1蛋白水平。 结果??与6、 12及48周比较， 48和72周雌鼠血清GSH-Px和T-AOC水平下降 （ P ?< 0.05） ，72 周 GSH-Px 与 48 周比较，差异有统计学意义（ P ?<0.05） ，48 周 T-AOC 与 12 周比较，差异有统计学 意义（ P ?<0.05） 。与 6、12 及 48 周比较，48 和 72 周雌鼠血清 FSH 和 LH 水平升高（ P ?<0.05） ，72 周 FSH 和 LH 水平与 48 周比较，差异有统计学意义（ P ?<0.05） 。48 和 72 周雌鼠血清 E 2 水平逐渐降低，12 和 24 周 E 2 水平与 48 周比较， 差异有统计学意义 （ P ?<0.05） ， 72 周 E 2 水平与各组比较， 差异有统计意义 （ P ?<0.05） 。随着时间的延长， 雌鼠卵巢 SIRT1 基因逐渐降低，48 和 72 周与 6、12 及 24 周 SIRT1?mRNA 比较，差异有统计学意义（ P ?<0.05） 。 48 和 72 周与 6 和 12 周 SIRT1 蛋白水平比较，差异有统计学意义（ P ?<0.05） ，72 周与 6、12 及 24 周 SIRT1 蛋白 水平比较，差异有统计学意义（ P ?<0.05） 。48 周雌鼠卵巢组织闭锁卵泡增多，黄体和各级发育卵泡减少，组织间 隙可见少量脂滴。72 周雌鼠卵巢组织闭锁卵泡明显增多， 黄体和各级发育卵泡明显减少， 卵泡颗粒细胞减少， 排 列松散， 组织间隙可见大量脂滴， 呈现明显衰老的特征。结论??衰老大鼠体重增加， GSH-Px 和 T-AOC 表达下降， 性激素分泌紊乱， 卵巢 SIRT1?mRNA 表达和蛋白水平降低， 卵巢明显退化。

Abstract:

Objective To investigate the expression changes of SIRT1 gene in ovary of aging female rats. Methods A total of 50 SD female rats were raised and sacrificed at 6, 12, 24, 48 and 72 weeks. Weigh were recorded every 3 days. The GSH-Px activity and T-AOC were determined by dithio dinitrobenzoic acid method and Fe 3+ reduction method, respectively. Serum levels of LH, FSH and E 2 were detected by ELISA. Morphological changes of ovarian tissue were observed by light microscopy. Expression of SIRT1 was detected by qPCR and immunohistochemistry. Results Serum levels of GSH-Px, T-AOC and E 2 in female rats were decreased significantly along with aging (P < 0.05), and statistically significant difference among various time point was observed in terms of GSH-Px, T-AOC and E 2 (P < 0.05). Circulating levels of FSH and LH were upregulated obviously along with aging (P < 0.05), and statistically significant difference among various time points were witnessed in terms of FSH and LH. With the prolongation of time, both protein and mRNA levels of SIRT1 gene in female ovarian were downregulated gradually , and statistical significance was obtained among various time points (P < 0.05). At the time point of 48 weeks, increased atresia follicles and decreased amount of luteal and developmental follicles as well as lipid droplets were observed in female ovarian tissue. Conclusions Aging ovary is manifested with decrease in GSH-Px and T-AOC as well as sexual hormone disorder, which may be mediated through downregulation of SIRT1 expression.