增殖细胞核抗原PCNA在大鼠缺血再灌注脑组织中的表达
[摘要] 目的 研究大鼠全脑缺血再灌注后增殖细胞核抗原PCNA的表达时程及其与凋亡的关系。方法 采用4-VO法建立全脑缺血再灌注损伤模型，将各组各时间点脑组织切片分别进行HE染色：光镜下观察海马回CA1区和CA3区神经元形态结构改变； 免疫组化染色：观察PCNA蛋白在CA1和CA3区的表达情况；TUNEL染色：观察海马回CA1区和CA3区神经细胞凋亡情况，统计阳性细胞表达情况，计算细胞凋亡指数；将PCNA蛋白免疫组化切片进行图象分析测得平均灰度值。结果 PCNA蛋白于再灌注后2h在海马CA1区可见表达下降，再灌注后6~24h组明显下降，之后持续低表达，CA3区表达弱于CA1区。缺血再灌注后6h凋亡细胞开始增加，主要位于CA1区，24h至48h为高峰，至72h明显减少。各时间点CA3区凋亡细胞数少于CA1区。两者的时间变化规律相似。结论PCNA蛋白主要在缺血再灌注后早期即24h前DNA损伤未积累到严重程度时执行抗凋亡的作用，对损伤神经元进行修复。
【关 键 词】脑缺血再灌注损伤；增殖细胞核抗原PCNA；DNA损伤；DNA修复；细胞凋亡
[Abstract] Objective To study the relationship of cell apoptosis with the expression of PCNA after the cerebral ischemia-reperfusion. Methods Global cerebral ischemia reperfusion model was produced by 4-VO method. All animals were executed at 2h, 6h, 12h, 24h, 48h, and 72h post-reperfusion by 4% poly-formaldehyde perfusion. HE staining, TUNEL staining and Immunohistochemical (IHC) staining of brain tissue section were performed at every time point. HE staining: The change of neuron structure in hippocampus were observed under light microscope. IHC staining: the expression of PCNA in CA1 and CA3 region of hippocampus were observed. TUNEL staining: The positive apoptosis nerve cells were observed in CA1 and CA3 region of hippocampus. TUNEL staining tissue section were observed under light microscope and calculated the apoptosis index. IHC staining tissue section were undertook image analysis to obtain the average gray scale numerical value. All the data were statistically analyzed . Results The expression of PCNA decreased in cell nucleus in CA1 region at 2h after cerebral ischemia reperfusion, the expression from 6h to 24h decreased deeply and decreased continuously after that, the expression in CA3 region was weaker than that in CA1 region. In IR group, it showed that apoptotic cell start increasing at 6h after cerebral ischemia reperfusion, mainly in CA1 region, reached apex from 24h to 48h, reduced obviously at 72h time point.; Expression in CA3 was weaker than that in CA1. Conclusion Proliferating cellnuclearantigen (PCNA) perform a protective function on neurons to fight against apoptosis and repair the neuronal DNA damage in earlier period after cerebral ischemia reperfusion.
[Key words] Cerebral ischemia/reperfusion injury; Proliferating cell nuclea ranti-gen (PCNA);DNA damage;DNA repair;Apoptosis
为了修补损伤、保证复制的真实性，生物体内有完善的机制来调控DNA复制到损伤修复的功能转换。那么，，PCNA在神经系统损伤后DNA修复中担任何种角色？许多研究者做了大量这方面的工作。发现PCNA在其中起了重要作用。Uberti等设计实验，用射线照射小鼠齿状回，发现PCNA参与损伤后的神经再生； Kaya等设计脑挫伤动物模型，检测PCNA在正常对照组动物细胞的表达，结果表明在胞浆内，检测脑挫伤48小时组的受损细胞的PCNA的表达，发现其转移到了细胞核内。Imai H等设计大脑中动脉阻塞模型，在缺血后2小时组中发现，缺血周边区只有很少PCNA免疫阳性细胞，在缺血后24小时组中，缺血周边区的PCNA免疫阳性细胞则明显增加。有研究者设计实验，用紫外线照射1周龄的小鼠海马脑片，造成海马细胞损伤，发现PCNA在CA3区锥体细胞中表达增高,该实验结果提示，PCNA在神经系统中非增生细胞中的表达与DNA的损伤修复密切相关[1]。国内徐广润等设计光化学法诱导老龄大鼠局灶性脑缺血实验，发现缺血后2小时，PCNA表达降低,但24h后在缺血周边区有所增强[2]。对大鼠进行缺血预处理后，PCNA阳性细胞在脑缺血区表达，与假手术组相比明显上调。提示在局灶性脑缺血再灌注过程中,PCNA参与了脑缺血后神经细胞对DNA损伤的修复过程, PCNA蛋白的表达变化可以影响DNA损伤的修复过程,受损神经元的功能恢复与DNA损伤的修复程度休戚相关。本研究主要观察大鼠在不同程度的脑缺血再灌注后，PCNA的表达改变极其与凋亡的时相与空间关系，探讨PCNA蛋白在脑缺血再灌注后DNA损伤修复中的作用。
2 结果
2.1 HE染色结果
2.2 TUNEL法检测细胞凋亡结果
3 讨论
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