缺氧缺血性脑损伤新生大鼠松果体钟基因Per1和Cry1表达的变化

Expressions of Perl and Cryl in the pineal gland of neonatal rats with hypoxic-ischemic brain damage

目的观察缺氧缺血性脑损伤（HIBD）新生大鼠松果体中PerlmRNA、CrylmRNA表达水平及PER1和CRY1蛋白合成水平的变化，探讨钟基因表达异常在HIBD导致的昼夜节律紊乱中可能扮演的重要角色。方法将7日龄新生sD大鼠72只，随机分为2组（HIBD组36只，对照组36只）。HIBD模型按改良Le-vine法建立。用半定量反转录（RT）．PCR和Westernblot法分别测定HIBD模型制备后0、2、12、24、36、48h2组新生大鼠松果体中PerlmRNA和CrylmRNA以及PER1、CRY1蛋白合成水平，并比较2组之间的差异。结果1．HIBD组PerlmRNA的表达水平在HIBD模型制备后24、36、48h均显著高于对照组（P均〈0．05），0、2、12h与对照组相比差异均无统计学意义（P均〉0．05）；HIBD模型制备后24hPerlmRNA水平开始升高，36h到达高峰，持续至48h。2．HIBD组CrylmRNA的表达水平在HIBD模型制备后12、24、36h均显著高于对照组（P均〈0．05），0、2、48h与对照组相比差异均无统计学意义（P均〉0．05）；HIBD模型制备后12hCrylmRNA水平开始升高，24h到达高峰，持续至36h。3．HIBD组PER1蛋白水平在HIBD模型制备后36h显著高于对照组（P〈0．05），0、2、12、24、48h与对照组相比差异均无统计学意义（P均〉0．05）。4．HIBD组CRY1蛋白水平在HIBD模型制备后2、12、24h均显著高于对照组（P均〈0．05），0、36、48h与对照组相比差异均无统计学意义（P均〉0．05）；HIBD模型制备后2hCRY1蛋白水平开始升高，24h到达高峰，36h降至正常。结论HIBD对新生大鼠松果体细胞中PerlmRNA、Cry1mRNA和PER1、CRY1蛋白水平均有显著影响，生物钟系统的紊乱可能与HIBD的发病有关。

Objective To explore the effects of clock genes on circadian disorder in hypoxic-ischemic brain damage（HIBD） by comparing the level of PER1, CRY1 synthesis and the expression of Perl mRNA, Cryl mRNA in pineal gland of neonatal rats with HIBD. Methods Seven-day-old Sprague-Dawley （SD） rats were randomly divided into 2 groups（ HIBD group and control group ,36 pups in each group）. HIBD models were set up according to modified Levine, euthanized 0,2,12,24,36,48 h afterwards. Semi-quantitative reverse transcriptase（RT） -PCR analysis was per- formed to measure the expression profiles of Perl mRNA and Cryl mRNA and Western blot was used to measure the PER1, CRY1 protein in rat pineal gland, and the differences between the 2 groups were compared. Results Compared with the control group ： 1. The expression of Perl mRNA in HIBD group displayed significantly higher than those of the control group at 24,36,48 h （ all P 〈 0.05 ）, while there were no statistically significant differences at 0,2,12 h （ all P 〉 0.05）. The levels of Perl mRNA in HIBD began to rise at 24 h, arrived to the peak at 36 h and remained till 48 h. 2. The expression of Cryl mRNA in HIBD group displayed significantly higher than those of the control group at 12,24, 36 h（ all P 〈 0. 05 ）, while there were no statistically significant differences at 0,2,48 h （all P 〉 0.05 ）. The levels of Cryl mRNA in HIBD began to rise at 12 h,arrived to the peak at 24 h and remained till 36 h. 3. The levels of PER1 protein in HIBD group surpassed those of the control group at 36 h （ P 〈 0.05 ）, and there were no statistically signifi- cant differences at 0,2,12,24,48 h（ all P 〉 0.05 ）. 4. The levels of CRY1 protein in HIBD group surpassed those of the control group at 12,24 and 36 h （all P 〈 0.05 ） , and there were no statistically significant differences at 0,2,48 h （all P 〉 0. 05 ）. The levels of CRY1 protein in HIBD began to rise at 2 h, arrived to the peak at 24 h and descended to nor- mal at 36 h. Conclusions HIBD indeed affects the level of PER1, CRY1 protein and the expression of Perl mRNA, Cryl mRNA in the neonatal rat pineal gland, while the disorder of biological clock may play important roles in the mechanism of HIBD.