海人酸诱导大鼠海马nestin的表达

为了探讨海人酸(kainicacid,KA)侧脑室注射后大鼠海马内nestin的表达变化,本文选用成年雄性SD大鼠,并随机分成实验组和对照组。实验组大鼠侧脑室注射1μl(0.5μg/μl)KA,分别于术后1、3、7、14、30、60、90d处死动物,并应用免疫荧光方法观察神经前体细胞标记物nestin、神经元特异烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)的表达。实验组大鼠海马的nestin阳性细胞在KA注射后3d开始出现于齿状回、CA3和CA1区,到7d达到高峰,然后逐渐减少。注射侧的nestin阳性细胞数均较非注射侧多(P<0.01);对照组仅有散在阳性细胞。本结果提示,海人酸可诱导和增强大鼠海马nestin的表达,而这些表达nestin的细胞主要为星形胶质细胞。     

海人酸对家兔海马和尾核神经元的作用

本文比较了成年兔和幼年兔神经元对海人酸的敏感性,并观察了海人酸对家兔海马和尾核神经元的作用。主要结果为: 1.成年兔侧脑室内注射不同剂量的海人酸后,海马不同部位的神经元有不同程度的损伤。注射剂量为0.8μg时,CA_3和CA_4区的大部分锥体细胞有明显的溃变,细胞皱缩,着色深,并有胶质细胞增生;同时尾核神经元也有严重损伤,胶质细胞大量增生。剂量为0.5μg或0.3μg时,CA_3和CA_4的锥体细胞约有1/2被破坏。剂量增至1μg时,锥体细胞损伤范围扩大,溃变更严重。 2.幼兔侧脑室内注射0.1μg或0.2μg海人酸后,海马的锥体细胞有一定的损伤,但仪限于CA_3a区。 3.向成年兔海马CA_2区注射0.3μg海人酸后,海马神经元即有明显的损伤,但只局限于注射的部位。成年兔尾核头部注射0.3μ海人酸后,可在注射部位观察到损伤的神经元和数在的胶质细胞。成年家兔海马锥体细胞对海人酸的敏感性较幼兔为强。海人酸对家兔海马锥体细胞的作用比较强,同时尾核神经元对海人酸也有一定的敏感性。     

海人酸致癫痫大鼠海马结构中noggin表达变化

目的:研究海人酸(kainic acid,KA)侧脑室注射并诱发癫痫持续状态(status epilepticus,SE)后大鼠海马结构中noggin基因在大鼠海马的表达变化。方法:大鼠在侧脑室注射KA后1、3、7、14、30和60d等不同时间,采用RT-PCR研究noggin mRNA含量变化,采用免疫组化观察noggin蛋白表达变化。结果:在正常大鼠海马结构中noggin mRNA有少量表达。noggin阳性细胞主要位于齿状回及CA3、CA1区,数量较少。侧脑室注射KA诱发SE后,noggin表达持续升高,3d达到高峰;7d在脑内的表达开始降低;注射后2个月,noggin表达降至术前水平,仅见散在的阳性细胞。结论:侧脑室注射KA并诱发SE后,大鼠海马结构中noggin表达明显增加。     

海人酸对神经干细胞增殖及分化的影响

背景：神经干细胞对脑组织的修复作用非常有限,约80%新增殖的内源性神经干细胞在6周内死亡,仅0.2%的细胞继续增殖、分化,参与修复。 目的：分析不同剂量海人酸在对神经干细胞增殖及分化的影响。 方法：体外分离并培养新生Wistar大鼠神经干细胞,将神经干细胞分为空白对照组和加入不同浓度梯度的海人酸组,通过免疫组化法和免疫荧光法进行鉴定,MTT比色法测定海人酸对神经干细胞分化的影响,计算分化后神经元和星形胶质细胞比例。 结果与结论：海人酸组贴壁的神经球分化速度较空白对照组快,在同一时间点进行观察,神经细胞的迁移距离较未处理组远。分化5 d后,海人酸组所分化的细胞中,星状细胞较空白对照组多,而神经元样细胞相对较少,培养的细胞具有自我更新和向神经元﹑少突胶质细胞和星形胶质细胞分化的潜能。兴奋性氨基酸海人酸可使部分神经干细胞死亡,但可促进幸存的神经干细胞增殖及分化,并诱导其向星形胶质细胞分化。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

癫痫大鼠海马内凋亡神经元超微结构的研究

目的　为揭示海人酸癫痫模型海马内神经元的死亡机制。　方法　选用海人酸诱导的癫痫大鼠模型,对海马内凋亡神经元的超微结构进行观察。　结果　电镜下,实验组大鼠海马齿状回门区和ＣＡ１区内可见散在的凋亡神经元,凋亡神经元主要表现为核周染色体的聚集和凝结成块,其核膜表现为皱缩和扭曲,在晚期凋亡的神经元有时可见核膜破裂。凋亡神经元胞浆内的细胞器保持完整。　结论　凋亡参与了海人酸诱发癫痫发作后海马内神经元的死亡过程。     

Par-4与阿尔茨海默病神经元凋亡

前列腺凋亡反应蛋白 4 (prostateapoptosisresponse 4 ,Par 4 )是一种小分子蛋白质 ,其生理功能尚不清楚 ,但能够促进多种细胞包括神经元的凋亡已得到证实。Par 4参与阿尔茨海默病 (Alzheimer’sdisease,AD)神经元凋亡过程复杂 ,且有望成为筛选治疗AD药物的作用靶点     

BMP4参与海人酸损伤后成年大鼠海马齿状回颗粒细胞增殖及胶质增生

目的探讨海人酸（KA）侧脑室注射致大鼠海马损伤后骨形成蛋白-4（BMP4）的表达变化及其与颗粒细胞增殖和胶质细胞增生的关系。方法将成年大鼠分为对照组与实验组。侧脑室注射KA7d后,用尼氏染色检测海马神经元丢失,用免疫组织化学与原位杂交的方法检测海马齿状回BMP4mRNA阳性细胞与BrdU标记细胞、GFAP阳性细胞数的变化。结果正常成年大鼠BMP4mRNA阳性细胞主要分布于海马齿状回的门区、颗粒下层、CA3、CAI区。BrdU标记细胞主要分布在齿状回颗粒下层。GFAP阳性细胞主要分布在齿状回、CA3区。在KA侧脑室注射致海马损伤后7d,海马CA3、CA4区神经元丢失明显,BMP4mRNA阳性细胞与BrdU、GFAP阳性细胞均明显增加。结论KA侧脑室注射致海马损伤后,成年大鼠海马齿状回颗粒细胞增殖增强和胶质增生可能与BMP4表达增加有关。     

嗜酸细胞Fas抗原表达及其在凋亡诱导中的作用

目的:探讨嗜酸细胞(Eos)凋亡基因Fas抗原的表达及其在凋亡诱导中的作用。方法:分离正常人外周血Eos,加入白细胞介素(IL)-5与不同浓度(1-1000μg/L)Fas单抗培养72h,观察Fas抗原表达、细胞存活和凋亡变化,并提取DNA电泳。结果:新分离的嗜酸细胞表达Fas抗原,Eos与单纯介质、IL-5培养后Fas抗原表达无明显变化。加入不同浓度的Fas单抗对IL-5介导的Eos存活均有显著抑制作用,当Fas单抗浓度为1000μg/L时,Eos存活率降为30%±12％(P＜0.01)。Fas单抗(100μg/L)处理,24h后,Eos即有明显的凋亡(35%±6%,P＜0.01)。提取DNA电泳呈典型凋亡梯状改变。结论:Fas抗原参与了Eos凋亡的调节,IL-5抑制Eos凋亡与Fas抗原表达水平无关。     

地塞米松对海人酸致（癇）大鼠脑P-糖蛋白表达的影响

目的：探讨地塞米松对海人酸致痫大鼠脑P-糖蛋白（P-gp）表达的影响.方法：将SD大鼠随机分为假手术组（Sham组,n=8）、癫痫组（EP组,n=12）、地塞米松干预癫痫组（DEX组,n=12）.后两组采用海马注射海人酸方法制作癫痫模型,DEX组癫痫造模前30 min给予腹腔注射地塞米松0.4 mg/kg.分别记录各组大鼠达到Ⅲ级和Ⅴ级发作时所需的时间（潜伏期）,初次至第6次≥Ⅳ级发作的间隔时间作为评价癫痫发作严重程度的指标;大鼠术后24 h处死,使用HE染色和免疫组织化学方法,比较各组海马CA3区、齿状回、杏仁核复合体区P-gp表达及脑损伤情况.结果：①Sham 组未见癫痫发作;DEX组与EP组达到Ⅲ级发作的潜伏期分别为（87.92±45.80）min和（67.50±22.91）min,达到Ⅴ级发作的潜伏期分别为（103.33±51.27 ）min和（75.60±22.10）min,差异均无统计学意义（P〉0.05）;与EP组相比,DEX组样发作严重程度降低（P=0.004）;②与EP组相比,DEX组于所观察的脑区损伤均减轻,以海马CA3区和杏仁核复合体区较为显著;③与Sham组比较,EP组各观察脑区P-gp表达均明显升高（P〈0.01）;与EP组相比,DEX组海马CA3区和杏仁核复合体区P-gp表达显著减少（P〈0.05）,而在齿状回表达量差异无统计学意义（P=0.078）.结论：地塞米松可降低海人酸致痫大鼠发作严重程度和脑损伤,抑制P-gp表达上调,其中以海马CA3区和杏仁核区较为显著.     

海人酸诱导成年大鼠颞叶癫痫中海马异常神经发生机制研究进展

神经发生(neurogenesis)是指神经干细胞(neural stem cells,NSCs)或神经前体细胞(neural precursor cells,NPCs)的增殖、分化以及新生神经元的存活、成熟和迁移整合的过程.实验证实~([1]),成年动物及人类的室管膜与室管膜下区、嗅球、海马、中隔、纹状体及皮质与脊髓广泛分布有多潜能的NSCs与NPCs,这些细胞能分化为有完整功能的神经元并整合入神经环路,提示中枢的神经发生不仅限于胚胎期,在生后乃至成年的整个发育期均能观察到神经发生.     

Overexpression of PDZ1 domain prevents apoptosis of rat hippocampal neurons induced by kainic acid

In our previous studies, Tat-GluR6-9c (a glutamate receptor 6 C-terminus peptide fused the TAT protein transduction sequence) not only inhibited the activation of MLK3 (mixed lineage kinase 3) and JNK (c-Jun N-terminal kinase) via the GluR6·PSD-95 (postsynaptic density protein 95)·MLK3 signaling module but also diminished neuronal death induced by kainic acid or transient cerebral ischemia in rat hippocampus. Here, we investigate whether overexpression of the PDZ1 domain of PSD-95 protein could suppress the binding of GluR6 with PSD-95 and the activation of MLK3, MKK7 (mitogen-activated kinase kinase 7) and JNK1/2, and rescused neuronal cell death induced by kainic acid. Our results showed that overexpression of the PDZ1 domain of PSD-95 protein could prevent nuclear accumulation and abrogate neuronal cell death in SD (Sprague–Dawley) rat hippocampal neuronal cells. Further studies indicated that overexpression of PDZ1 could inhibit the enhancement of binding of GluR6 to PSD-95 and prevent the activation of MLK3, MKK7 and JNK1/2 induced by kainic acid. Taken together, the essential role of the PDZ1 domain of PSD-95 in apoptotic cell death in neurons provides an experimental foundation for gene therapy of neurodegenerative diseases with overexpression of the PDZ1 domain.     

阿司匹林通过抑制EphA4通路保护缺血造成的脑白质损伤

目的:我们前期研究发现阿司匹林具有脑白质保护作用,但其机制尚不清楚。有研究报道Eph信号与白质损伤密切相关,为此本研究拟探讨大鼠脑白质损伤后阿司匹林对Eph受体表达变化的影响。方法:成年雄性SD大鼠利用双侧永久性颈总动脉结扎模型造成脑白质损伤(WML)。随机分为正常组、WML组和阿司匹林处理组(每日100 mg/kg,ip,30d),每组15只。采用Real-time PCR法检测正常组胼胝体内8种Eph受体的表达情况;免疫荧光染色检测Eph受体是否表达在少突胶质细胞中;Western Blot检测各组Eph受体和髓鞘碱性蛋白(myelin basic protein,MBP)的表达变化及相互关系。结果:Real-time PCR结果:8种Eph受体中,EphA4的mRNA表达水平最高。EphA4与CNPase的免疫荧光染色示:EphA4表达于胼胝体内的少突胶质细胞。与正常组相比,WML组中EphA4~+细胞数量增多(P0.05),阿司匹林处理后EphA4~+细胞数量减少(P0.05)。Western Blot结果显示:与正常组比较,WML组胼胝体部EphA4表达升高(P0.05),但MBP蛋白表达下降(P0.05);阿司匹林处理后,可下调EphA4(P0.05)、上调MBP的表达水平(P0.05),二者呈显著负相关。结论:阿司匹林可能通过抑制EphA4通路保护缺血造成的脑白质损伤。     

烟碱拮抗秋水仙碱诱导的乳大鼠大脑皮质神经元凋亡

目的：探讨烟碱拮抗秋水仙碱诱导乳大鼠大脑皮质神经元凋亡的作用机制。方法: 培养Sprague-Dawley（SD）乳大鼠大脑皮层神经元，Hoechst33258核荧光染色进行凋亡率计数检测神经元凋亡, Akt（ser473）Western blotting分析试剂盒做激酶分析。结果: 0.1 μmol/L秋水仙碱可降低皮层神经元的存活率，诱导大鼠皮层神经元凋亡。但是，当10 μmol/Ｌ烟碱预孵2 h后再加入0.1 μmol/L秋水仙碱作用24 h, 烟碱可拮抗秋水仙碱的此种凋亡作用,将凋亡率从（62±1）％ 降低到 (38±2) ％,P<0.05。Akt（ser473）磷酸化在0.5 h时开始下降，于6 h时最低，仅为对照组的1/3。且Akt（ser473）磷酸化水平随时间（0.5、1、6、12和18 h）呈钟型性改变,分别是对照组的1.3、3.7、2.4、2.1和1.9倍,P<0.05。结论: 烟碱对大鼠皮质神经元凋亡的拮抗作用可能与其激活Akt有关。     

Lower and reduced expression of EphA4 is associated with advanced TNM stage,lymph node metastasis,and poor survival in breast carcinoma

The expression of EphA4 has been well documented in the development of nerve and in certain types of human cancer. Few studies of EphA4, however, have focused on breast carcinoma. In this study, a set of breast carcinomas was subjected to immunohistochemical staining. In normal luminal cells, EphA4 was weakly detected in 11 (14.3 %), moderately detected in 15 (19.5 %) and highly detected in 51 out of 77 (66.2 %) samples, while in breast carcinoma cells, EphA4 was weakly detected in 42 (54.5 %), moderately detected in 19 (24.7 %) and highly detected in 16 out of 77 (20.8 %) samples (P < 0.001). The expression of EphA4 protein was significantly reduced in 68.8 % of breast carcinoma samples comparing with normal cells. The expression of EphA4 was significantly associated with tumor grade (P = 0.003), TNM stage (P = 0.034), lymph node metastasis (P = 0.034) and Ki‐67 (P < 0.001). No significant relationship was found between the expression of EphA4 and age, molecular subtypes, and HER2 status. Survival analysis showed that significant association of low expression of EphA4 in tumor cells with short overall survival (P = 0.048) and disease‐free survival (P = 0.051). Our data show that EphA4 was reduced in breast carcinoma, which is associated with high grade, advanced TNM stage, lymph node metastasis, and poor outcome of patients.     

红藻氨酸诱导PC12细胞凋亡及阿魏酸对神经元的保护作用

 目的： 探讨阿魏酸(ferulic acid, FA)对红藻氨酸(kainic acid, KA)诱导的PC12细胞凋亡的作用及其机制。方法：采用50 μmol/L KA诱导PC12细胞凋亡建立阿尔茨海默病神经细胞模型,然后将处理后的PC12细胞分为KA模型组和KA＋FA (25、50和100 μmol/L)处理的低、中、高剂量组,同时设立正常对照组。采用MTT比色法检测PC12细胞的存活率;采用免疫细胞化学法观察PC12细胞中凋亡蛋白Bcl-2、Bax和细胞色素C (Cyt C)的表达;annexin Ⅴ+PI双染流式细胞术检测PC12的细胞凋亡率;蛋白免疫印记技术检测PC12细胞中Bcl-2、Bax和Cyt C的表达水平。结果：MTT法和免疫细胞化学检测显示,与正常组相比,模型组PC12细胞的存活率明显下降,且细胞中Bcl-2表达减少(P<0.01),而Bax和Cyt C表达升高,Bcl-2/Bax比值下降(P<0.01),流式细胞术检测细胞的凋亡率显示,模型组细胞的凋亡率显著上升(P<0.01)。蛋白印迹术检测显示,模型组细胞中Bcl-2表达量减少,Bax和Cyt C表达量升高,与正常组比较差异显著(均P<0.01)。当采用FA干预后,与模型组相比,25、50和100 μmol/L组细胞的存活率明显上升,细胞凋亡率减少,而且能增加Bcl-2阳性百分率和表达水平,明显减少Bax和Cyt C阳性百分率和表达水平,使Bcl-2/Bax比值增加 (P<0.05或P<0.01)。结论：KA在50 μmol/L时可明显诱导PC12发生凋亡,FA在25～100 μmol/L时能显著抑制KA诱导的PC12细胞凋亡,其神经保护机制可能是通过抑制Bax和Cyt C的表达,升高Bcl-2表达和Bcl-2/Bax比值,从而阻断内源性细胞凋亡通路而提高神经细胞的存活率。     

红藻氨酸对大鼠海马锥体细胞钙电流的影响

目的：应用膜片钳技术,观察红藻氨酸（KA）对大鼠海马锥体细胞ca2＋电流的影响,以研究癫痫的发病机制。方法：采用酶加机械分离法制备出生10～12d的大鼠海马锥体神经元标本,用全细胞膜片钳技术测定其生理学特性及观察KA对ca2＋电流的影响。结果：分离出的海马锥体细胞形态正常,有较长突起;用膜片钳技术证实,其保存了主要的离子通道活性。KA20μmol／L和100μmol／L的浓度均可使海马锥体细胞ca2＋电流峰值增大（n=8,P〈0．01）。结论：①大鼠海马锥体神经细胞具有明显的突起,细胞膜表面光洁、晕光好,适用于膜片钳实验研究;②KA使ca2＋内流增加,引起“Ca”超载”导致细胞毒性等一系列反应;③KA通过激活α-氨基羟甲基恶唑丙酸（AMPA）受体,诱发快速的兴奋性突触后电位（EPSP）,参与兴奋性突触传递。AMPA受体的激活可能是癫痫的发病机制之一。     

Inhibition of EphA4 signaling after ischemia-reperfusion reduces apoptosis of CA1 pyramidal neurons

The purinergic receptors P2X₄ and P2X₆ are ion channels activated by ATP. These receptors are present in the gastrointestinal tract, and they are involved in synaptic transmission, taste sensation, and pain, among other functions. In this work, we studied the distribution of P2X₄ and P2X₆ receptors in proximal and distal regions of the gut newborn and adult rats. Using immunohistochemistry, purinergic receptors were found in gut epithelial cells and capillary vessels. In both proximal and distal regions of newborn rats, we observed P2X₄ signal in epithelial cells, whereas P2X₆ was present in capillary vessels in the proximal region and to a lesser extent in the distal region. In both regions of adult gut, we observed P2X₄ and P2X₆ immunostain in the capillary vessels. Semi-quantification indicated a significant difference in the amount of P2X₄ between proximal regions, whereas the P2X₆ content of both newborn regions differed from that in adult proximal gut. We conclude that P2X₄ and P2X₆ purinoreceptors are present in the gut from birth and that they are differentially distributed among regions.     

Expressions of EphA2 and EphrinA-1 in early squamous cell cervical carcinomas and their relation to prognosis

By using immunohistochemistry we investigated the expression of EphA2 and EphrinA-1 in 217 early squamous cell cervical carcinomas and examine their prognostic relevance. For EphA2 expression, 21 tumors (10%) showed negative, 108 (50%) weak positive, 69 (32%) moderate positive and 19 (9%) strong positive, whereas for EphrinA-1 expression, 33 tumors (15%) showed negative, 91 (42%) weak positive, 67 (31%) moderate positive and 26 (12%) strong positive. In univariate analysis high expression (strong staining) of EphrinA-1 was associated with poor disease-free (P = 0.033) and disease-specific (P = 0.039) survival. However, in the multivariate analyses neither EphrinA-1 nor EphA2 was significantly associated to survival. The increased levels of EphA2 and EphrinA-1 in a relative high number of early stage squamous cell carcinomas suggested that these two proteins may play an important role in the development of a subset of early cervical cancers. However, EphA2 and EphrinA-1 were not independently associated with clinical outcome.