[摘要] 目的  研究大鼠全腦缺血再灌注后增殖細(xì)胞核抗原PCNA的表達(dá)時(shí)程及其與凋亡的關(guān)系。方法 采用4-VO法建立全腦缺血再灌注損傷模型，將各組各時(shí)間點(diǎn)腦組織切片分別進(jìn)行HE染色：光鏡下觀察海馬回CA1區(qū)和CA3區(qū)神經(jīng)元形態(tài)結(jié)構(gòu)改變； 免疫組化染色：觀察PCNA蛋白在CA1和CA3區(qū)的表達(dá)情況；TUNEL染色：觀察海馬回CA1區(qū)和CA3區(qū)神經(jīng)細(xì)胞凋亡情況，統(tǒng)計(jì)陽性細(xì)胞表達(dá)情況，計(jì)算細(xì)胞凋亡指數(shù)；將PCNA蛋白免疫組化切片進(jìn)行圖象分析測得平均灰度值。結(jié)果 PCNA蛋白于再灌注后2h在海馬CA1區(qū)可見表達(dá)下降，再灌注后6~24h組明顯下降，之后持續(xù)低表達(dá)，CA3區(qū)表達(dá)弱于CA1區(qū)。缺血再灌注后6h凋亡細(xì)胞開始增加，主要位于CA1區(qū)，24h至48h為高峰，至72h明顯減少。各時(shí)間點(diǎn)CA3區(qū)凋亡細(xì)胞數(shù)少于CA1區(qū)。兩者的時(shí)間變化規(guī)律相似。結(jié)論P(yáng)CNA蛋白主要在缺血再灌注后早期即24h前DNA損傷未積累到嚴(yán)重程度時(shí)執(zhí)行抗凋亡的作用，對損傷神經(jīng)元進(jìn)行修復(fù)。
【關(guān) 鍵 詞】腦缺血再灌注損傷；增殖細(xì)胞核抗原PCNA；DNA損傷；DNA修復(fù)；細(xì)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

對腦缺血后神經(jīng)元損傷機(jī)制研究中，DNA損傷和DNA修復(fù)是近年來的熱點(diǎn)。很多研究者發(fā)現(xiàn)，腦缺血后不僅發(fā)生神經(jīng)細(xì)胞的壞死，在缺血周圍,也即所謂半暗帶區(qū)可產(chǎn)生DNA損傷，由此可以誘導(dǎo)凋亡。
為了修補(bǔ)損傷、保證復(fù)制的真實(shí)性，生物體內(nèi)有完善的機(jī)制來調(diào)控DNA復(fù)制到損傷修復(fù)的功能轉(zhuǎn)換。那么，，PCNA在神經(jīng)系統(tǒng)損傷后DNA修復(fù)中擔(dān)任何種角色？許多研究者做了大量這方面的工作。發(fā)現(xiàn)PCNA在其中起了重要作用。Uberti等設(shè)計(jì)實(shí)驗(yàn)，用射線照射小鼠齒狀回，發(fā)現(xiàn)PCNA參與損傷后的神經(jīng)再生； Kaya等設(shè)計(jì)腦挫傷動(dòng)物模型，檢測PCNA在正常對照組動(dòng)物細(xì)胞的表達(dá)，結(jié)果表明在胞漿內(nèi)，檢測腦挫傷48小時(shí)組的受損細(xì)胞的PCNA的表達(dá)，發(fā)現(xiàn)其轉(zhuǎn)移到了細(xì)胞核內(nèi)。Imai H等設(shè)計(jì)大腦中動(dòng)脈阻塞模型，在缺血后2小時(shí)組中發(fā)現(xiàn)，缺血周邊區(qū)只有很少PCNA免疫陽性細(xì)胞，在缺血后24小時(shí)組中，缺血周邊區(qū)的PCNA免疫陽性細(xì)胞則明顯增加。有研究者設(shè)計(jì)實(shí)驗(yàn)，用紫外線照射1周齡的小鼠海馬腦片，造成海馬細(xì)胞損傷，發(fā)現(xiàn)PCNA在CA3區(qū)錐體細(xì)胞中表達(dá)增高,該實(shí)驗(yàn)結(jié)果提示，PCNA在神經(jīng)系統(tǒng)中非增生細(xì)胞中的表達(dá)與DNA的損傷修復(fù)密切相關(guān)[1]。國內(nèi)徐廣潤等設(shè)計(jì)光化學(xué)法誘導(dǎo)老齡大鼠局灶性腦缺血實(shí)驗(yàn)，發(fā)現(xiàn)缺血后2小時(shí)，PCNA表達(dá)降低,但24h后在缺血周邊區(qū)有所增強(qiáng)[2]。對大鼠進(jìn)行缺血預(yù)處理后，PCNA陽性細(xì)胞在腦缺血區(qū)表達(dá)，與假手術(shù)組相比明顯上調(diào)。提示在局灶性腦缺血再灌注過程中,PCNA參與了腦缺血后神經(jīng)細(xì)胞對DNA損傷的修復(fù)過程, PCNA蛋白的表達(dá)變化可以影響DNA損傷的修復(fù)過程,受損神經(jīng)元的功能恢復(fù)與DNA損傷的修復(fù)程度休戚相關(guān)。本研究主要觀察大鼠在不同程度的腦缺血再灌注后，PCNA的表達(dá)改變極其與凋亡的時(shí)相與空間關(guān)系，探討PCNA蛋白在腦缺血再灌注后DNA損傷修復(fù)中的作用。

1.實(shí)驗(yàn)方法
2 結(jié)果
2.1  HE染色結(jié)果
2.2  TUNEL法檢測細(xì)胞凋亡結(jié)果 
3 討論

參 考 文 獻(xiàn)

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