大鼠局灶性脑缺血再灌注后海马Bcl-2、Bax蛋白的表达

目的 探讨大鼠局灶性脑缺血再灌注后Bcl-2、Bax蛋白在海马表达的变化.方法 线栓法制作大鼠局灶性脑缺血再灌注模型,应用免疫组化染色检测Bcl-2、Bax蛋白表达,应用TUNEL法检测海马区细胞凋亡.结果 缺血再灌注2h后海马神经元Bcl-2、Bax蛋白开始表达,Bcl-2蛋白12h达高峰,Bax蛋白12h～24h达高峰,之后开始下降.再灌注2h后海马凋亡细胞开始表达,随着再灌注时间的延长,其表达不断增加.Bcl-2/ Bax的比率在再灌注开始时升高,再灌注12h达高峰,随后开始下降.结论 凋亡是脑缺血再灌注损伤的重要形式之一,Bcl-2/ Bax的改变与缺血再灌注后海马的神经元存亡有关,缺血再灌注可导致海马神经元凋亡.     

bFGF对大鼠局灶性脑缺血后神经细胞凋亡及Bcl-2、Bax的影响

目的探讨bFGF对缺血后神经细胞的保护作用。方法用线栓法制作局灶性脑缺血大鼠模型,于术前1h、术后第1天、第2天连续3天侧脑室注射bFGF,分1μg／d、2μg／d、4μg／d3组,观察缺血程度、梗塞体积、Bcl-2、Bax蛋白的合成。结果;bFGF能减轻脑缺血程度,减少梗塞体积(25．2％)及凋亡细胞数,提高半暗带内Bcl-2蛋白的合成,减低缺血灶内Bax蛋白的合成,各剂量组间无显著差异。结论bFGF可作为一种有效的神经细胞保护剂,保护神经细胞免受缺血的损害。     

大鼠全脑缺血再灌注损伤后调控基因Bcl-2、Bax的表达及与细胞凋亡的关系

目的:本研究旨在探讨Bcl-2及Bax蛋白在大鼠全脑缺血再灌注损伤中的变化及与细胞凋亡的关系。方法:雄性Wistar大鼠56只,随机分为假手术组、缺血15分钟再灌注1、6、12、24、48、72小时组。采用大鼠四条血管阻断方法制备大鼠全脑缺血再灌注模型。采用TUNEL法观察不同再灌注时间组海马CAl区细胞凋亡的变化。采用免疫组化法观察Bcl-2及Bax蛋白表达水平的变化。结果:脑缺血损伤后随再灌注时间延长凋亡细胞逐渐增多,至再灌注48小时达到高峰,72小时后减少。Bcl-2表达至再灌注12小时达高峰,再灌注24~72小时组逐渐减弱。Bax表达至48小时达高峰,再灌注72小时减少。结论:Bcl-2于再灌注早期表达增强,Bax于再灌注中期表达增强,Bcl-2/Bax比例失衡可能是大鼠全脑缺血再灌注后神经细胞凋亡的机制之一。     

阿司匹林预处理对大鼠局灶性脑缺血再灌注后神经细胞凋亡和Bcl-2、Bax蛋白表达的影响

目的观察预先使用阿司匹林(ASA)进行药物处理对大鼠局灶性脑缺血/再灌注(CI/RP)损伤后神经细胞凋亡及其调节基因(Bcl-2、Bax)表达的影响。方法48只SD雄性大鼠被随机分为对照组(n=12)和实验组(n=36),实验组又分为:小剂量组(ASA10 mg/kg)(n=12)、中剂量组(ASA50mg/kg)(n=12)、大剂量组(ASA150mg/kg)(n=12)。实验组于术前5d连续给予相应剂量的ASA灌胃。然后建立大鼠局部脑缺血2h、再灌24h的动物模型。第6天处死各组大鼠,TTC染色法测定脑梗死体积,TUNEL技术原位标记凋亡细胞,免疫组化法检测凋亡调节基因相关蛋白Bcl-2和Bax的表达。结果ASA干预后,小、中、大剂量ASA组梗死病灶体积与对照组相比均减小,缺血周边区凋亡细胞阳性率均显著降低。小、中剂量ASA组的作用优于大剂量ASA组,小,中剂量两个实验组缺血区域Bcl-2蛋白的表达增加,Bax蛋白的表达降低,3个实验组间进行比较,小、中剂量ASA组的作用优于大剂量ASA组。结论ASA预处理可以缩小大鼠脑缺血再灌注损伤后的梗死体积,可以减少大鼠缺血再灌注损伤脑组织的细胞凋亡,这一过程可能与上调Bcl-2蛋白的表达和减少Bax蛋白的表达有关,抑制CI/RP后细胞凋亡的表达可能是ASA的神经保护作用之所在。     

bFGF对大鼠局灶性脑缺血后神经细胞凋亡及外Bcl-2、Bax的影响

目的：探讨bFGF对缺血后神经细胞的保护作用．方法：用线栓法制作局灶性日缺血大鼠模型,于术前1h、术后第1天、第 2天连续3天侧脑室注射bFGF,分 1μg／d、2μg／d、4 μg／d 3组,观察缺血程度、梗塞体积、Bcl-2、Bax蛋白的合成。结果：bFGF能减轻脑缺血程度,减少梗塞体积（25.2％）及凋亡细胞数,提高半暗带内Bcl-2蛋白的合成,减低缺血灶内Bax蛋白的合成,各剂量组间无显著差异。结论：bFGF可作为一种有效的神经细胞保护剂．保护神经细胞免受缺血的损害。     

大鼠脑缺血再灌注后Caspase-3、Bcl-2和Bax的表达

目的探讨大鼠脑缺血再灌注后caspase-3、Bcl-2和Bax在脑皮质神经元中的表达。方法将动物随机分为假手术组及缺血组,参照zea longa线栓法建立大鼠左侧大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)局灶性脑缺血再灌注模型,各组大鼠分别在左侧MCAO2h再灌注不同时间点断头取脑,脑皮质神经元中caspase-3、Bcl-2和Bax的表达通过免疫组化法来测定。结果缺血组大鼠脑皮质caspase-3的表达较假手术组显著增强(P<0.01),缺血组大鼠脑皮质Bcl-2的表达较假手术组显著增强(P<0.01),缺血组大鼠脑皮质Bax的表达较假手术组显著增强(P<0.01)。结论短暂性脑缺血再灌注上调脑皮质神经元中caspase-3和Bax的表达促细胞凋亡,上调脑皮质神经元中Bcl-2的表达抗细胞凋亡。     

高血压大鼠局灶性脑缺血再灌注后Survivin及Bcl-2表达

目的:研究肾性高血压大鼠局灶性脑缺血再灌注后脑组织Survivin、Bcl- 2表达和细胞凋亡,探讨其意义。方法:体重2 90～3 10g肾性高血压Wistar大鼠5 0只随机分成2组:A组(缺血再灌注组)及B组(假手术组)。采用线栓法制作局灶性脑缺血2h再灌注模型,假手术组不造成缺血。用免疫组化法和TUNEL法分别观察再灌注6、12、2 4、48和72h脑组织Survivin、Bcl- 2表达和凋亡细胞。结果:在缺血再灌注组中:①再灌注6h时较多Survivin表达,到72h仍见强烈表达;②再灌注6h即可见Bcl -2表达较多,2 4h达高峰,此后逐渐下降;③再灌注6h后已出现较多凋亡细胞,在72h达高峰。假手术组及再灌注组的非缺血侧未见Survivin、Bcl- 2阳性细胞,但见0～2个凋亡细胞。结论:局灶性脑缺血再灌注后Survivin、Bcl- 2表达显著增高,细胞凋亡参与了脑缺血再灌注过程。     

大鼠局灶性脑缺血Bcl-2和Bax的表达与细胞凋亡

目的:研究大鼠局灶性脑缺血再灌注后凋亡相关基因Bcl-2和Bax在缺血皮层表达的变化及其与神经元凋亡的关系。方法:线栓法制作大鼠局灶性脑缺血再灌注模型,免疫组化法观察Bcl-2和Bax的表达变化,TUNEL法观察神经元凋亡的情况。结果:再灌注2h后皮层神经元Bcl-2表达开始明显上调,6h为高峰,之后开始下降。再灌注早期 Bax在皮层神经元的表达即明显增强,24～48h达高峰。Bcl-2／Bax的比率在再灌注开始时升高,6h达高峰,随后开始下降。TUNEL阳性细胞主要分布在缺血中心的边缘,再灌注48h之内,随时间的延长而不断增加。结论:Bcl-2／Bax的比率改变与缺血再灌注后的神经元存亡相关。     

大鼠脑缺血再灌注后Bcl-2、Fas蛋白的表达及意义

目的　探讨 Ｂｃｌ２ 及 Ｆａｓ 蛋白在大鼠脑缺血再灌注损伤中的表达及与缺血性凋亡的关系。方法　采用免疫组化方法观察 Ｂｃｌ２ 及 Ｆａｓ 蛋白在脑缺血再灌注后随时间延长动态变化并用图像分析测定二者的免疫强度。结果　脑缺血再灌注后 Ｂｃｌ２、 Ｆａｓ 表达。 Ｂｃｌ２ 蛋白表达于再灌注 ３ｈ 达高峰,再灌注 ６ｈ 其表达呈下降趋势,再灌注 ２４ｈ 仅少数细胞阳性表达。 Ｆａｓ 蛋白表达于再灌注 ６ｈ 达高峰,对缺血较敏感的海马大锥体细胞亦有表达,再灌注 ２４ｈ 其表达减少。结论　 Ｆａｓ 蛋白表达介导了脑缺血后细胞凋亡的发生,并可能参与了迟发性神经元死亡。 Ｂｃｌ２ 表达与神经元存活密切相关,在神经元缺血敏感性方面起重要作用,对神经元起保护作用。     

大鼠脑缺血再灌注损伤后的细胞凋亡与Bcl-2、Bax的表达

目的:观察脑缺血再灌注损伤中的细胞凋亡与Bcl2家族的关系。方法:参照ZeaLonga线栓法制作急性大鼠大脑中动脉缺血再灌注模型,观察脑缺血再灌注后大鼠缺血脑组织中的凋亡细胞变化情况及Bcl2、Bax蛋白表达情况,同时通过透射电镜观察缺血侧脑组织神经元超微结构变化。结果:同对照组相比缺血再灌注组凋亡神经元细胞增多(TUNEL阳性细胞)(P<0.05);Bax、Bcl2阳性细胞均升高(P<0.01),同时Bcl2/Bax比值降低;电镜观察神经元超微结构出现凋亡早期改变。结论:Bcl2/Bax比值影响脑缺血再灌注损伤后细胞凋亡。     

脂肪来源神经干细胞移植局灶性脑缺血模型大鼠的血管新生

背景：脂肪组织来源的神经干细胞移植可改善脑缺血大鼠神经功能,但其机制尚不明确。 目的：观察人脂肪组织来源神经干细胞移植对大鼠局灶性脑缺血后血管新生的影响。 方法：体外培养脂肪基质细胞,诱导分化为神经干细胞。60只健康雄性SD大鼠分为4组：正常组6只,假手术组6只,缺血对照组24只,移植治疗组24只。后两组线栓法制作大鼠大脑中动脉缺血2 h再灌注模型,又分为缺血2 h再灌注7,14,21,28 d组,每个时点各6只。假手术组不闭塞大脑中动脉。造模成功后24 h,移植治疗组经尾静脉移植人脂肪组织来源神经干细胞悬液,细胞浓度为2×109 L-1;缺血对照组经尾静脉注射生理盐水。免疫组织化学法进行微血管密度计数,观察脑缺血区血管增生情况。 结果与结论：免疫组织化学结果显示,与缺血对照组比较,移植治疗组缺血2 h再灌注7,14,21,28 d的微血管密度值均显著高于缺血对照组,差异有显著性意义(P < 0.05~0.01)。提示脂肪组织来源的神经干细胞移植可促进脑缺血区新生血管的形成。     

3-硝基丙酸预处理对大鼠局灶性脑缺血Bcl-2和Bax mRNA表达的影响

目的探讨3-硝基丙酸(3-NPA)预处理对大鼠局灶性脑缺血半暗带Bc l-2和Bax mRNA表达的影响。方法将大鼠腹腔注射3-NPA 20 mg/kg,3 d后制作局灶性脑缺血再灌注模型;采用逆转录聚合酶链反应,观察3-NPA预处理对脑缺血再灌注1 h、6 h、12 h、24 h及48 h额顶部皮质Bc l-2和Bax mRNA表达的影响,并与假手术组和缺血再灌注组比较。结果与假手术组比较,缺血再灌注组和3-NPA预处理组各时间点Bc l-2和Bax mRNA表达极显著增强(均P<0.01);与缺血再灌注组比较,3-NPA预处理组各时间点Bc l-2mRNA表达显著增强(均P<0.05),再灌注12~48 h Bax mRNA的表达显著降低(均P<0.05)。结论增强Bc l-2的表达、抑制Bax的表达,可能是3-NPA预处理抑制细胞凋亡、诱导脑缺血耐受的机制之一。     

Anti-apoptotic effects of aspirin following cerebral ischemia-reperfusion injury in rats☆

BACKGROUND： The pharmacological effects of aspirin on apoptosis are complex. The underlying mechanisms have not been properly defined. OBJECTIVE： To observe the effect of different doses of aspirin on brain cell apoptosis following focal cerebral iscbemia-reperfusion injury （CIRI） in rats. DESING, TIME AND SETTING： A randomized, controlled, animal experiment, performed at the School of Medicine and Pharmaceutics, Jiangnan University between June and October 2006. MATERIALS： Twenty-six male, adult, Sprague Dawley rats （grade Ⅱ）, weighing 240-290 g, were obtained from Shanghai Experimental Animal Center, Chinese Academy of Sciences. Aspirin was provided by Sigma （USA）. METHODS： The rats were randomly divided into four groups： sham-operation （SO）, CIRI ＋ vehicle, CIRI ＋ aspirin （6 mg/kg）, and CIRI ＋ aspirin （60 mg/kg）. Rats in the lesion groups were intragastrically administrated saline, aspirin （6 mg/kg）, or aspirin （60 mg/kg）, respectively. MAIN OUTCOME MEASURES： The number of pyramidal neurons with normal appearance in the cerebral cortex at 24 mm from the midline; apoptotic cell death as measured by TUNEL; Bcl-2 and Bax protein localization was determined by immunohistochemistry; malondialdehyde （MDA） and super oxidation （SOD） content were determined by biochemistry method; adenosine triphosphate （ATP） content measured by capillary electrophoresis. RESULTS： Following CIRI, the following parameters were altered compared with sham-operated animals： the number of neurons with normal appearance was significantly reduced in the cerebral cortex; the number of apoptotic cells increased; Bax protein expression was enhanced; and the ratio between Bcl-2 and Bax decreased. In addition, MDA content increased significantly, whereas ATP content decreased （P 〈 0.01）. Aspirin ameliorated the loss of healthy pyramidal neurons. Both 6 and 60 mg/kg aspirin increased the ratio between Bcl-2 and Bax, with no significant difference between the treatment group     

Anti-apoptotic effects of aspirin following cerebral ischemia-reperfusion injury in rats

BACKGROUND: The pharmacological effects of aspirin on apoptosis are complex. The underlying mechanisms have not been properly defined. OBJECTIVE: To observe the effect of different doses of aspirin on brain cell apoptosis following focal cerebral ischemia-reperfusion injury (CIRI) in rats. DESING, TIME AND SETTING: A randomized, controlled, animal experiment, performed at the School of Medicine and Pharmaceutics, Jiangnan University between June and October 2006. MATERIALS: Twenty-six male, adult, Sprague Dawley rats (grade II), weighing 240-290 g, were obtained from Shanghai Experimental Animal Center, Chinese Academy of Sciences. Aspirin was provided by Sigma (USA). METHODS: The rats were randomly divided into four groups: sham-operation (SO), CIRI vehicle, CIRI aspirin (6 mg/kg), and CIRI aspirin (60 mg/kg). Rats in the lesion groups were intragastrically administrated saline, aspirin (6 mg/kg), or aspirin (60 mg/kg), respectively. MAIN OUTCOME MEASURES: The number of pyramidal neurons with normal appearance in the cerebral cortex at 2-4 mm from the midline; apoptotic cell death as measured by TUNEL; Bcl-2 and Bax protein localization was determined by immunohistochemistry; maiondiaidehyde (MDA) and super oxidation (SOD) content were determined by biochemistry method; adenosine triphosphate (ATP) content measured by capillary electrophoresis. RESULTS: Following CIRI, the following parameters were altered compared with sham-operated animals: the number of neurons with normal appearance was significantly reduced in the cerebral cortex; the number of apoptotic cells increased; Bax protein expression was enhanced; and the ratio between Bcl-2 and Bax decreased. In addition, MDA content increased significantly, whereas ATP content decreased (P < 0.01 ). Aspirin ameliorated the loss of healthy pyramidal neurons. Both 6 and 60 mg/kg aspirin increased the ratio between Bcl-2 and Bax, with no significant difference between the treatment groups. In addition, 60 mg/kg aspirin decreased MDA content and increased ATP levels. However, 6 mg/kg aspirin did not have the same effect. CONCLUSION: Aspirin reduced the number of apoptotic cells following CIRI. These results suggest that the neuroprotective mechanism of aspirin could be related to elevated Bcl-2 protein levels or decreased Bax protein expression. The increase in the ratio of Bcl-2 to Bax appears to be a common anti-apoptotic mechanism of aspirin.     

脑出血患者血肿周围组织神经元凋亡与Bcl-2、Bax蛋白表达关系的研究

目的探讨脑出血(ICH)患者血肿周围组织神经元凋亡与凋亡相关基因Bcl2、Bax蛋白表达的关系。方法采用缺口末端标记法、免疫组化法分别检测ICH患者血肿周围组织神经元凋亡率和Bcl2、Bax表达水平,分析神经元凋亡率与Bcl2、Bax表达及Bax/Bcl2值的关系;出血量与Bcl2、Bax表达及Bax/Bcl2值的关系,以及神经元凋亡率与出血量、病程、神经功能缺损程度评分(NDS)的关系。结果ICH患者血肿周围组织神经元凋亡率及Bcl2、Bax表达明显高于正常对照组(均P<0.01);血肿周围组织神经元凋亡率与Bcl2表达呈负相关(r=-0.682,P<0.01),与Bax、Bax/Bcl2值表达呈正相关(r=0.592、0.740,均P<0.01)。出血量与血肿周围组织Bcl2表达呈负相关(r=-0.677,P<0.01),与Bax表达及Bax/Bcl2值呈正相关(r=0.654、0.751,均P<0.01)。细胞凋亡率与出血量及NDS呈正相关(r=0.829、0.897,均P<0.01),与病程不相关。结论细胞凋亡机制参与了ICH后继发性神经元损伤;Bcl2、Bax蛋白及Bax/Bcl2值对凋亡具有调控作用。     

奥扎格雷钠对大鼠局灶性脑缺血血小板内皮细胞黏附分子-1、Bcl-2、Bax表达的影响

目的 观察大鼠局灶性脑缺血后脑组织中血小板内皮细胞黏附分子-1(PECAM-1、CD31)、Bcl-2、Bax表达的改变及奥扎格雷钠对其表达的影响. 方法 采用线栓法制作大鼠大脑中动脉局灶性脑缺血模型,制模成功大鼠随机分为奥扎格雷钠组和生理盐水组,脑组织切片免疫组化染色检测不同时间点PECAM-1、Bcl-2、Bax在各组大鼠脑组织中的表达变化. 结果大鼠大脑中动脉闭塞后脑组织PECAM-1、Bcl-2、Bax的表达明显增高(均P＜0.01).奥扎格雷钠组缺血12h、24h、36h表达PECAM-1较生理盐水组增高,缺血12h、24h 表达Bcl-2较生理盐水组增高,缺血24h、36h表达Bax较生理盐水组增高,均P＜0.01. 结论 脑组织表达的PECAM-1、Bcl-2、Bax分别参与了脑缺血不同时期的病理生理作用;奥扎格雷钠可促进PECAM-1和Bcl-2的表达,抑制Bax的表达.     

Stem cell transplantation for treatment of cerebral ischemia in rats Effects of human umbilical cord blood stem cells and human neural stem cells

BACKGROUND:Exogenous neural stem cell transplantation promotes neural regeneration. However, various types of stem cells transplantation outcomes remain controversial. OBJECTIVE:To explore distribution, proliferation and differentiation of human neural stem cells (hNSCs) and human umbilical cord blood stem cells (hUCBSCs) following transplantation in ischemic brain tissue of rats, and to compare therapeutic outcomes between hNSCs and hUCBSCs. DESIGN, TIME AND SETTING:Randomized controlled animal studies were performed at the Experimental Animal Center of Nanjing Medical University and Central Laboratory of Second Affiliated Hospital of Nanjing Medical University of China from September 2008 to April 2009. MATERIALS:hNSCs were harvested from brain tissue of 10-13 week old fetuses following spontaneous abortion, and hUCBSCs were collected from umbilical cord blood of full-term newborns at the Second Affiliated Hospital of Nanjing Medical University of China. hNSCs and hUCBSCs were labeled by 5-bromodeoxyuridine (BrdU) prior to transplantation. METHODS:Rat models of cerebral ischemia were established by the suture method. A total of 60 healthy male Sprague Dawley rats aged 7-9 weeks were randomly assigned to hNSC transplantation, hUCBSC transplantation and control groups. The rat models in the hNSC transplantation, hUCBSC transplantation and control groups were infused with hNSC suspension, hUCBSC suspension and saline via the caudal vein, respectively. MAIN OUTCOME MEASURES:The distribution, proliferation and differentiation of hNSCs and hUCBSCs in ischemic brain tissue were observed using immunohistochemical methods. Neurological function in rats was assessed using the neurological severity score. RESULTS:The number of BrdU-positive cells was significantly greater in the hNSC transplantation group compared with hUCBSC transplantation group at 14 days following transplantation (P < 0.05). The number of BrdU-positive cells reached a peak at 28 days following transplantation. Nestin-positive, glial fibrillary acidic protein-positive, cyclic nucleotide 3' phosphohydrolase-positive and neuron specific enolase-positive cells were visible following transplantation. No significant difference was determined in the constituent ratio of various cells between hNSC and hUCBSC transplantation groups (P > 0.05). The neurological severity score was significantly decreased in rats at 21 days following transplantation (P < 0.05). No significant difference was detected in neurological severity score between hNSC and hUCBSC transplantation groups at various time points (P > 0.05). CONCLUSION:The transplanted hNSCs and hUCBSCs can migrate into ischemic brain tissue, proliferate and differentiate into neuron-like, astrocyte-like and oligodendrocyte-like cells, and improve neurological function in rats with cerebral ischemia.