摘要
目的观察缺血再灌注损伤（IRI）对大鼠肝组织内细胞周期调控相关基因转录和表达的影响，探讨生长阻滞和DNA损伤修复基因（Gadd45a）和早期生长反应基因1（Egr-1）参与DNA损伤后修复的可能机制。方法按随机成组对照设计将大鼠平均分为正常对照组、缺血组、再灌注30min、1h、2h、3h及24h组，于相应时间点获取大鼠肝组织样本，将利用芯片实验筛选出的关键基因Gadd45a和Egr-1作为目的基因，并采用RNA印迹法和实时荧光定量逆转录聚合酶链反应（RT-PCR）验证基因芯片筛查的结果，采用荧光免疫组织化学染色检测冰冻新鲜肝组织样本Egr-1的表达，采用免疫组织化学染色检测肝组织样本Gadd45a的表达。结果RNA印迹检测结果显示，RT-PCR产物与待测的目标基因一致；表达谱芯片信号与RT-PCR的检测显示，目的基因的表达变化特征均为再灌注早期（1h）升高4倍以上，其中Egr-1的表达上调9．39倍，Gadd45a的表达上调8．28倍及热休克蛋白70—1a的表达上调10．8倍；再灌注后24h均降至正常水平，芯片初筛结果与RT-PCR的检测结果一致，是准确、可靠的。荧光免疫组织化学检测显示，再灌注1h组和2h组肝组织细胞核Egr-1的表达均为阳性；免疫组织化学检测显示，随着再灌注时间的增加，细胞核和细胞浆内Gadd45的表达逐渐升高。结论Gadd45a和Egr-1基因均是核因子κB调控通路的关键基因，在大鼠肝脏IRI中参与调控了细胞周期G2／M期的停滞和修复受损的DNA。
Objective To explore the possible mechanism that Gadd45a and Egr-1 together participate in the DNA damage repair, by analyzing the influence of hepatic ischemia-reperfusion injury （IRI） on the mRNA and protein expression of hepatic cell cycle-related genes. Method Methods Forty-two SD rats were divided into normal control group, ischemia 30 rain group and reperfusion 30 rain, 1 h, 2 h, 3 h and 24 h groups. The hepatic specimens of rats at the corresponding time points after success of modeling were obtained. Chip experiment was used to screen out the key genes Gadd45a, Egr-I and GAPDH for target genes. We used Northern blot and RT-PCR to confirm the chiKs results. The expression of related proteins was detected by using immunofluorescenee and immunohistochemistry. Results Northern blot detection results showed that the RT-PCR products and the target genes for testing were concordant; The expression spectral chip signal and RT-PCR detection revealed that the expression of target genes had a 4-fold increase at early reperfusion period （1 h） （up-regulation of Egr-1, Gadd45a and HSP70-la by 9. 39, 8.28 and 10. 8 respectively. At the later stage of reperfusion （24 h）, the expression of Egr-1, Gadd45a and Hspala was reduced to the normal levels, and the chip screening results were consistent with the RT-PCR test results. Fluorescence immunohistochemistry demonstrated that in the reperfusion 1 h and 2 h groups, the expression of Egr-1 in nuclei of liver cells was positive. Immunohistochemistry indicated that the Gadd45 expression in the nucleus and cytoplasm was gradually increased with increasing reperfusion time. Conclusion Gadd45a and Egr-1 are the key genes of the nuclear factor （NF）-κB regulatory pathways, and involved in the regulation of ceil cycle arrest at G2/M phase and repair of damaged DNA in rat hepatic IRI.
出处
《中华器官移植杂志》
CAS
CSCD
北大核心
2012年第8期466-469,共4页
Chinese Journal of Organ Transplantation
基金
湖南省自然科学基金（09JJ3061）
美国中华医学会CMB基金（06837）
