高血压大鼠全脑缺血再灌注后神经细胞DNA损伤修复的研究

目的 从DNA损伤和修复的角度探讨局灶性脑缺血再灌注后神经元凋亡的机制.方法 运用双肾双夹法建立肾血管性高血压大鼠模型,采用四血管法制备高血压大鼠全脑缺血再灌注模型,免疫组化法检测再灌注过程中鼠脑DNA损伤修复,蛋白XRCC1、TUNEL法检测凋亡.结果 与假手术组相比,脑缺血再灌注3h在缺血区皮质和海马CA1区XRCC1表达出现显著减低,这种减低一直持续到缺血再灌注24h(P＜0.05);再灌注24h缺血区皮质和海马CA1区神经细胞发生了凋亡,神经细胞XRCC1的表达强度与凋亡呈负相关.结论 脑缺血再灌注早期神经细胞XRCC1表达的减少可能导致机体对此时出现的DNA单链断裂修复的能力下降,造成了再灌注后期发生了凋亡.     

GM1对高血压大鼠脑缺血再灌注后DNA损伤修复的影响

目的探讨GM1对局灶性脑缺血再灌注后神经元凋亡作用的机制。方法运用双肾双夹法建立肾血管性高血压大鼠模型，采用线栓法制备高血压大鼠短暂性局灶性脑缺血再灌注模型，免疫组化法检测再灌注过程中鼠脑DNA损伤修复蛋白XR—CC1、TUNEL法检测凋亡。结果与假手术组相比，脑缺血再灌注3h至24h在缺血区皮质XRCC1表达出现显著减低（P〈0.05）；再灌注24h缺血区皮质神经细胞发生凋亡，神经细胞XRCC1的表达强度与凋亡呈负相关。GM1治疗后缺血区皮质XRCC1的表达显著升高，凋亡显著减少（P〈0．05）。结论GM1通过提高肾血管性高血压大鼠局灶性脑缺血再灌注后神经细胞XRCC1的表达，发挥了抗神经细胞凋亡的作用。     

大鼠局灶性脑缺血再灌注ICRmRNA表达动态变化研究

目的 探讨白细胞介素1-β转化酶（ICE）在局灶性脑缺血再灌注后的表达及作用。方法 线栓法复制大脑中动脉脑缺血再灌注模型。逆转录聚合酶链式反应（RT-PCR）技术检测大鼠局灶性脑缺血再灌注后ICEmRNA表达。结果 缺血3h及缺血3h再灌注随缺血及缺血再灌注时间延长,缺血中心区与半影区ICEmRNA表达处于动态变化之中,再灌注24h、48h半影区表达持续高水平,而中心区表达下降。结论 局灶性脑缺血再灌注过程中ICEmRNA表达增强,促进神经细胞凋亡,ICE参与局灶性脑缺血再灌注神经细胞凋亡的调控。     

用逆转录-多聚酶链反应检测局灶脑缺血及再灌注后c-fos和c-jun基因表达

本研究应用逆转录－多聚酶链反应（ＲＴ－ＰＣＲ）方法检测大鼠局灶脑缺血模型中即早基因ｃ－ｆｏｓ和ｃ－ｊｕｎ的表达。结果发现缺血１５ｍｉｎ时可见到ｃ－ｆｏｓ和ｃ－ｊｕｎｍＲＮＡ表达缺血３０ｍｉｎ时引起轻微左侧局灶脑缺血改变，可诱导左侧局灶脑缺血区ｃ－ｆｏｓ和ｃ－ｊｕｎｍＲＮＡ广泛的表达；缺血９０ｍｉｎ后，导致大面积局灶脑缺血改变，诱导上述两种基因在同侧缺血区与同侧非大脑中动脉（ＭＣＡ）供血区的海马中表达。后者有相当轻的缺血症状。再灌流６０ｍｉｎ后诱导两种基因的共同表达立即达高峰。我们采用标准化的大鼠局灶脑缺血及再灌注模型，在分子水平上动态观察缺血／再灌注后基因变化特征，为缺血性脑损害的防治提供实验依据。     

西洛他唑对大鼠脑缺血再灌注损伤后PARP/AIF介导的细胞凋亡途径的影响

目的 研究大鼠局灶性脑缺血再灌注损伤后多聚腺苷二磷酸核糖聚合酶(PARP)和凋亡诱导因子(AIF)在海马CAI区的表达,探讨西洛他唑预处理能否通过PARP/AIF途径发挥脑保护作用. 方法 将135只雄性SD大鼠按随机数字表法分为3组:假手术组、模型组、西洛他唑组,每组45只.采用线栓法阻塞大鼠大脑中动脉制作局灶性脑缺血再灌注损伤模型.西洛他唑组造模前灌胃给予30 mg/kg剂量西洛他唑(2次).每组根据再灌注时间点不同(6 h、24 h、72 h)分为3个亚组,每亚组15只.应用TUNEL法检测神经细胞凋亡变化,Western blotting法检测AIF、腺苷二磷酸核糖(PAR)在不同时间点的变化,RT-PCR法检测AIF mRNA的表达变化. 结果 大鼠局灶性脑缺血再灌注损伤后出现AIF核移位.与假手术组比较,模型组凋亡细胞数明显增加,AIF、PAR含量及AIF mRNA表达明显增加,24 h时最显著,差异均有统计学意义(P＜0.05).西洛他唑组再灌注6h、24 h、72 h各亚组凋亡细胞数较模型组中各亚组明显减少,AIF、PAR含量较模型组各亚组明显降低,AIF mRNA表达亦明显减少,24h时最显著,差异有统计学意义(P＜0.05). 结论 西洛他唑对大鼠脑缺血再灌注损伤有一定保护作用,其抗神经细胞凋亡的机制之一可能是通过抑制脑缺血损伤引起的PARP的过度活化及AIF的易位而实现的.     

大鼠脑缺血再灌注损伤后NF-κB蛋白、Caspase3 mRNA表达及细胞凋亡的研究

目的　研究大鼠局灶性脑缺血再灌注损伤后核蛋白因子 κB(NF κB)蛋白、天冬氨酸特异性半胱氨酸蛋白酶 3(Caspase3)mRNA表达及细胞凋亡的变化。方法　采用大脑中动脉线栓法复制局灶性脑缺血再灌注模型 ,应用免疫组化、原位杂交法检测NF κB蛋白、Caspase3mRNA的表达 ,利用原位缺口末端标记法 (TUNEL)研究凋亡细胞的变化。结果　与假手术大鼠相比 ,脑缺血再灌注后NF κB蛋白、Caspase3mRNA表达明显增强 ;凋亡细胞显著增多 (P <0 .0 1)。结论　大鼠局灶性脑缺血再灌注损伤引发的神经细胞凋亡可能是与上调NF κB蛋白、Caspase3mR NA表达有关。     

吸氧预处理对大鼠局灶性脑缺血再灌注损伤后神经细胞凋亡及CHOP/GADD153表达研究

目的研究吸氧预处理(oxygen inhalating precondition,OIP)对大鼠局灶脑缺血再灌注后内质网应激反应(Indoplasmic recticulum stress,ERS)导致的细胞凋亡及凋亡相关基因生长停滞及DNA损伤基因153(C/EBPhomologous protein/growth arrest and DNA damage-inducible gene 153,CHOP/GADD153)的关系,探讨OIP的脑保护作用。方法采用线栓法阻塞大鼠大脑中动脉制作局灶脑缺血再灌注模型:(1)Tunel法标记神经细胞凋亡;(2)免疫组化染色法和western-blot法观察GADD153蛋白含量。结果与模型组比较,OIP组海马CA1区的凋亡细胞率明显下降(P<0.05),GADD153阳性细胞平均灰度值和蛋白含量相对值均明显下降(P<0.05)。结论 OIP可通过抑制缺血再灌注后神经细胞凋亡和减少GADD153蛋白的表达发挥脑保护作用。     

大鼠脑缺血再灌注后内质网应激相关因子表达的改变

目的观察内质网应激相关因子GRP78和CHOP在大鼠局灶性脑缺血再灌注后的表达,探讨内质网应激在脑缺血再灌注损伤中的作用。方法采用大鼠大脑中动脉线栓模型,应用免疫组化及RT-PCR方法检测脑缺血再灌注后不同时相缺血周围区GRP78和CHOP的表达;缺口末端标记法检测细胞凋亡。结果模型组GRP78和CHOP表达均高于假手术组,分别于再灌注12h和24h达高峰,CHOP表达与神经细胞凋亡时间趋势一致。结论脑缺血再灌注诱导GRP78和GADD153表达上调,与神经细胞凋亡有关,内质网应激机制参与了脑缺血再灌注后的神经细胞凋亡。     

JAK2/STAT3信号转导通路在缺血性脑损伤中作用机制的研究

目的 探讨磷酸化JAK2、磷酸化STAT3蛋白在局灶性脑缺血再灌注损伤后的表达及JAK2-STAT3信号转导通路在缺血性脑损伤中的作用.方法 采用大脑中动脉线栓法制作大鼠局灶性脑缺血再灌注损伤模型,应用Western blotting检测大鼠局灶性脑缺血再灌注损伤后磷酸化JAK2、磷酸化STAT3蛋白表达水平的变化.利用原位缺口末端标记法(TUNEL法)研究神经细胞凋亡的变化.同时应用JAK2特异性抑制剂AG490观察其影响.结果 大鼠局灶性脑缺血再灌注损伤后,再灌注损伤3h P-JAK2、P-STAT3蛋白可见少量表达,12h表达增强,24h达高峰,以后逐渐下降,168h后仍有少量表达.缺血再灌注损伤后凋亡细胞也显著增多,再灌注24～48h达高峰,凋亡细胞的变化与磷酸化JAK2、磷酸化STAT3蛋白表达变化一致.应用JAK2特异性抑制剂AG490不但阻断JAK2的磷酸化、STAT3的酪氨酸磷酸化,而且具有抗细胞凋亡作用.结论 脑缺血再灌注损伤后可引发JAK2、STAT3的活化,JAK2/STAT3信号通路可能参与了脑缺血再灌注损伤机制.     

吸氧预处理对大鼠局灶脑缺血神经细胞凋亡及Bcl-2、Bax的影响

目的 观察吸氧预处理对大鼠局灶脑缺血再灌注后细胞凋亡及凋亡相关基因Bax、Bcl-2的关系,探讨吸氧预处理的脑保护作用.方法 采用线栓法阻塞大鼠大脑中动脉造成局灶脑缺血再灌注模型,用TUNEL染色标记神经细胞凋亡,免疫组化染色观察Bax,Bcl-2蛋白含量.结果 与模型组相比,吸氧预处理组半暗区的TUNEL阳性细胞数和Bax蛋白表达阳性细胞数明显下降,(P<0.05),Bcl-2蛋白表达细胞数明显上升(P<0.05).结论 吸氧预处理可通过抑制缺血再灌注后神经细胞凋亡和减少Bax的表达,增加Bcl-2蛋白的表达发挥脑保护作用.     

Effects of transient global ischemia and kainate on poly(ADP-ribose) polymerase (PARP) gene expression and proteolytic cleavage in gerbil and rat brains

Poly (ADP-ribose) polymerase (PARP) is involved in various cellular functions, including DNA repair, the cell cycle and cell death. While PARP activation could play a critical role in repairing ischemic brain damage, PARP inactivation caused by caspase 3-cleavage may also be important for apoptotic execution. In this study we investigated the effects of transient global ischemia and kainic acid (KA) neurotoxicity, in gerbil and rat brains, respectively, on PARP gene expression and protein cleavage. PARP mRNA increased in the dentate gyrus of gerbil brains 4 h after 10 min of global ischemia, which returned to basal levels 8 h after ischemia. KA injection (10 mg/kg) also induced a marked elevation in PARP mRNA level selectively in the dentate gyrus of rat brains 1 h following the injection, which returned to basal levels 4 h after the injection. These observations provide the first evidence of altered PARP gene expression in brains subjected to ischemic and excitotoxic insults. Using both monoclonal and polyclonal antibodies to PARP cleavage products, little evidence of significant PARP cleavage was found in gerbil brains within the first 3 days after 10 min of global ischemia. In addition, there was little evidence of significant PARP cleavage in rat brains within 2 days after kainate (KA) injection. Though these findings show that caspase induced PARP cleavage is not substantially activated by global ischemia and excitotoxicity in whole brain, the PARP mRNA induction could suggest a role for PARP in repairing DNA following brain injury.     

Role of superoxide in poly(ADP-ribose) polymerase upregulation after transient cerebral ischemia

Oxidative stress plays a pivotal role in ischemic-reperfusion cell injury. Oxygen-derived free radicals trigger DNA strand damage, which is responsible for the activation of poly(ADP-ribose) polymerase (PARP). Recent studies have shown that peroxynitrite is the primary mediator of DNA damage and, hence, PARP activation after ischemia. PARP activation depletes NAD and ATP pools, ultimately resulting in necrotic cell death by loss of energy stores. Our study shows that PARP is upregulated as early as 15 min after 1 h of transient focal cerebral ischemia and remains for 8 h. We also examined the role of superoxide in PARP induction using copper/zinc-superoxide dismutase transgenic mice. Immunohistochemical and Western blotting data showed that there was no increased induction in PARP expression in these mice, suggesting that one of the mechanisms by which ischemic injury is attenuated in these mice might be by the inhibition of PARP induction. Furthermore, double staining of ischemic tissue with a PARP antibody and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) indicated that most cells that are positive for TUNEL do not stain for the PARP antibody, confirming recent reports that PARP activation is involved in necrotic cell death rather than apoptosis during ischemic-reperfusion injury.     

Apoptosis and protein expression after focal cerebral ischemia in rat

We used double staining histochemistry to investigate the relationship between apoptotic cell death and selective protein expression associated with DNA damage (p53, Bax, MDM2, Gadd45), DNA repair (PCNA) and cell cycle proteins (cyclin A, cyclin D, cdk2, cdk4) in rats (n=6; control rats, n=5) subjected to transient (2 h) middle cerebral artery occlusion (MCAo) and 46 h of reperfusion. Few apoptotic cells were detected in the non-ischemic hemisphere of control rats. In ischemic animals, scattered apoptotic cells were present in the ischemic core and clustered apoptotic cells were present and localized to the inner boundary zone of the ischemic core. Proteins were preferentially localized to the cellular cytoplasm of control rats and in the non-ischemic hemisphere of rats subjected to MCAo. However, after MCAo these proteins were expressed and were preferentially localized to nuclei within the ischemic lesion. DNA damage induced proteins (wt-p53 and p53-response proteins) were preferentially expressed within apoptotic cells after ischemia. DNA repair proteins and cell cycle proteins were preferentially expressed within morphologically intact cells and in reversibly damaged cells in the ischemic areas. The selective expression of proteins associated with DNA damage, DNA repair and cell cycle observed in morphologically intact cells, ischemic injured cells and apoptotic cells suggests a differential role for these proteins in cell survival and apoptosis after stroke.     

老龄大鼠局灶性脑缺血后DNA修复蛋白：增生细胞核抗原表达的研究

目的：探讨增生细胞核抗原（Ｐｒｏｌｉｆｅｒａｔｉｇｎ ｃｅｌｌ ｎｕｃｌｅａｒ ａｎｔｉｇｅｂｎＰＣＮＡ）在老龄大鼠局灶性脑缺血后不同时空表达改变的规律。方法：采用２６月龄Ｗｉｓｔａｒ大鼠,在光化学诱导局灶性脑缺血后,应用免疫组织化学和原位杂交的方法从蛋白质和ｍＲＮＡ水平观察不同时间和空间状态下ＰＣＮＡ表达。结果：缺血４小时缺血周边区有少许核染色为棕褐色颗粒的神经元和大量的胶质细胞;缺血２４小时在     

大鼠局灶性脑缺血再灌注损伤后STAT1蛋白表达的研究

目的探讨ＳＴＡＴ１基因在大鼠局灶性脑缺血再灌注损伤中的表达及其在缺血性神经元损伤中可能的作用。方法采用线栓法制作大鼠局灶性脑缺血再灌注损伤模型，用免疫组化方法观察ＳＴＡＴ１蛋白在大鼠局灶性脑缺血再灌注损伤后不同时间点脑组织中的表达。结果ＳＴＡＴ１蛋白在脑缺血６０ｍｉｎ再灌注２４ｈ后呈阳性表达，半暗带损伤区表达最显著，表达持续时间达１周。结论ＳＴＡＴ１蛋白超量表达可能对神经细胞存活和修复过程是有益的     

大鼠局灶性脑缺血预处理的抗细胞凋亡作用机制的研究

目的研究大鼠短暂局灶性脑缺血预处理对再次脑缺血神经细胞凋亡的保护作用,及bcl-2、bax与脑缺血耐受的关系.方法用开颅方法阻断大鼠大脑中动脉(MCA)20分钟,3天后再次阻断6小时.观察大鼠脑梗死体积及组织病理学改变,采用TUNEL法观察神经细胞凋亡状况,采用免疫组织化学方法观察bcl-2、bax蛋白表达的改变.结果与假预处理组和缺血组相比,预处理后缺血组梗死灶体积明显减小(均P＜0.01),半影区凋亡细胞数明显减少(P＜0.01),bax蛋白表达下降(P＜0.05),bcl-2蛋白表达显著上升(P＜0.01).结论 20分钟局灶性脑缺血预处理能够通过bcl-2表达增加及bax表达下降对再次脑缺血神经细胞起保护作用.     

Early decrease of apurinic/apyrimidinic endonuclease expression after transient focal cerebral ischemia in mice.

The authors examined the protein expression of apurinic/apyrimidinic endonuclease (APE/Ref-1), a multifunctional protein in the DNA base excision repair pathway, before and after transient focal ischemia in mice. Immunohistochemistry showed the nuclear expression of APE/Ref-1 in the entire region of the control brains. Nuclear immunoreactivity was decreased as early as 5 minutes after 60 minutes of ischemia in the ischemic core, which was followed by a significant reduction of APE/Ref-1-positive cells in the entire middle cerebral artery territory. Western blot analysis of the sample from the nonischemic brain showed a characteristic 37-kDa band, which was reduced after ischemia. A significant amount of DNA fragmentation was observed at 24 hours, but not at 4 hours, after ischemia. The authors' data provide the first evidence that APE/Ref-1 rapidly decreases after transient focal ischemia, and that this reduction precedes the peak of DNA fragmentation in the brain regions that are destined to show necrosis and apoptosis. Although further examination is necessary to elucidate the direct relationship between the APE/Ref-1 decrease and ischemic necrosis and apoptosis, our results suggest the possibility that rapid decrease of APE/Ref-1 and the failure of the DNA repair mechanism may contribute to necrosis or apoptosis after transient focal ischemia.     

大鼠局灶性脑缺血再灌注后血管内皮细胞凋亡及其与P53表达的关系

目的 探讨大鼠局灶性脑缺血再灌注损伤后血管内皮细胞凋亡及其与P53蛋白表达的关系。方法 采用原位末端标记法和免疫组化法分别观察脑缺血再灌注2、6、12小时,1、2、3、7、14和21天血管内皮细胞凋亡和P53蛋白表达的变化。结果 脑缺血再灌注2小时在缺血周围区即有凋亡的内皮细胞出现,12－24小时达高峰,之后逐渐下降,至21天与假手术组已无显著性差异。脑缺血再灌注6小时在缺血周围区P53蛋白开始表达,1－2天达高峰,之后逐渐下降,至7天与假手术组已无显著性差异。P53蛋白表达高峰时间迟于内皮细胞凋亡24小时。结论 脑缺血再灌注损伤中凋亡是血管内皮细胞的死亡形式之一,P53蛋白表达参与缺血再灌注后血管内皮细胞凋亡机制的调节。     

神经节苷脂对大鼠脑缺血再灌注损伤的脑保护作用

目的探讨神经节苷脂对大鼠脑缺血再灌注损伤的脑保护作用。方法采用线栓法制作缺血再灌注大鼠模型,分别用神经节苷脂(治疗组)和生理盐水(对照组)腹腔注射。观察两组大鼠缺血90min、缺血90min再灌注24h的脑梗死面积、神经功能缺损程度、细胞凋亡数、细胞凋亡率。结果治疗组大鼠于相同时间点脑梗死面积较对照组明显减小,仅表现轻度的神经功能缺损,且神经细胞的凋亡数较对照组显著减少(均P<0.01)。结论神经节苷脂能明显减小大鼠实验性脑缺血的脑梗死面积,减轻脑缺血再灌注后神经功能缺损程度,显著减轻缺血区神经元损害,具有显著的脑保护作用。     

亚低温对大鼠局灶性脑缺血再灌注后多聚腺苷二磷酸核糖聚合酶表达的影响

目的研究亚低温对大鼠局灶性脑缺血再灌注后多聚腺苷二磷酸核糖聚合酶(PARP-1)不同时空表达的影响,进一步探讨亚低温脑保护作用的分子机制。方法线栓法建立大鼠大脑中动脉阻塞再灌注模型,分假手术组、假手术 亚低温组、模型组及模型 亚低温组。应用Western blotting和免疫组化技术分别检测再灌注后不同时相缺血侧皮层PARP-1蛋白的表达与裂解。结果模型组PARP-1蛋白表达量随再灌注时间的延长逐渐增加,至再灌注24h达高峰,然后逐渐减少,再灌注72h时仍高于假手术组的水平;模型组PARP-1蛋白出现裂解,随再灌注时间的延长,裂解逐渐增强。每一相同再灌注时间点,模型 亚低温组PARP-1蛋白表达量和裂解片段含量均低于模型组。结论PARP-1的过度表达和裂解是局灶性脑缺血再灌注神经元死亡的重要分子机制。亚低温可通过抑制PARP-1的过度表达及减少PARP-1的裂解而发挥脑保护作用。