三七总皂苷抗缺氧缺糖再给氧诱导大鼠海马神经细胞凋亡的研究

目的 :以原代培养的大鼠海马神经细胞缺氧 /缺糖再给氧为模型 ,观察三七总皂苷对神经细胞凋亡的抑制作用。方法 :流式细胞仪检测凋亡细胞百分率 ,激光共聚焦扫描显微镜检测细胞内Ca²⁺浓度 ,荧光显微镜观察细胞形态学变化和坏死细胞百分率 ,同时 ,测定细胞乳酸脱氢酶 (LDH)的释放。结果 :神经细胞缺氧 /缺糖 5h后再给氧可诱导神经细胞凋亡和细胞坏死 ,并显著增加细胞内Ca²⁺浓度和LDH的释放 ,并随再给氧时间的延长而增加。三七总皂苷能降低神经细胞凋亡及坏死的百分率 ,降低细胞内Ca²⁺浓度 ,减少LDH的释放 ,三七总皂苷的作用随剂量增加而作用增强。结论 :三七总皂苷对缺血再灌注损伤后海马神经元的凋亡过程具有抑制作用 ,这种作用可能与其能降低细胞内Ca²⁺浓度有关。     

同时检测两种运动神经元存活基因第7外显子缺失的简便方法及其临床应用

目的：建立同时检测运动神经元存活基因SMNT和SMNC第7外显子缺失的简便方法，用于儿童期起病的脊髓性肌萎缩症（SMA）的临床诊断。方法：应用聚合酶链反应（PCR）－限制性酶切技术，用一对引物同时扩增55例正常成年人和5例SMA患儿及其父母的SMN基因2个成员的第7外显子中的高度保守区，扩增产物经限制性内切酶酶切及非变性聚丙烯酰胺凝胶电泳。结果：此法可检测2个SMN基因第7外显子的缺失情况，经检测，55例正常成人均无SMNT基因第7外显子缺失，SMNC基因第7外显子缺失仅3例，5例SMA患儿的SMNT基因第7外显子均有缺失，患儿父母无SMNT基因和SMNC基因第7外显子缺失，结论：此法对检测2个SMN基因第7外显子的缺失提供了简便，特异，且适合临床特别是基层医院应用，优于PCRSSCP分析的方法。     

灯盏花中单体成分对体外培养大鼠视网膜神经细胞的影响

目的:考察咖啡酸、东莨菪内酯、野黄芩苷对体外培养视网膜神经细胞存活的影响,探讨灯盏花中具视神经保护作用的有效成分。方法:采用胰酶消化法将18只出生2-3d的乳鼠视网膜制成细胞悬液,接种于经多聚鸟氨酸(HA)和层粘连蛋白(LN)包被的96孔板中。于培养3d后,分别加入PBS液及不同含量的咖啡酸、东莨菪内酯、野黄芩苷溶液继续培养2d,采用MTT比色法测量其存活细胞的吸收值,同时对部分细胞行Nissel体染色检查。结果:Nissel体染色检查结果表明,培养5d的存活细胞大部分均为神经细胞。与空白对照组比较,咖啡酸含量在3.9-1000μg·mL^-1、东莨菪内酯、野黄芩苷含量在250-1000μg·mL^-1时,其吸收值均明显增加(P<0.05,P<0.01),且有一定的量效关系。结论:咖啡酸、东莨菪内酯、野黄芩苷三者均能促进体外培养的视网膜神经细胞存活,且咖啡酸活性最强。     

Excitotoxic profile of LY339434, a GluR5 agonist, in cultured murine cortical neurons

The neurotoxic profile of (2S,4R, 6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434), a low-affinity kainate receptor subtype 5 (GluR5) agonist at recombinant human glutamate receptors, was evaluated to investigate the involvement of GluR5 in excitotoxic neuronal death. Murine cortical neurons were exposed to treatments for 24 h and assessed by a cell viability assay and phase-contrast microscopy. LY339434 (1-1000 microM) caused a concentration-dependent decrease in cell viability (EC(50)=11.4+/-1.2 microM) that was only attenuated by (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine (MK-801, 10 microM), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 microM) or 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466, 20 microM). Labeling with nucleic acid binding dyes revealed that LY339434 induced few apoptotic-like characteristics. These findings indicate that in cultured murine cortical neurons, LY339434 acts predominantly through N-methyl-D-aspartate (NMDA) receptors rather than GluR5 to effect neuronal death that is rapid and involves predominantly necrosis rather than morphological apoptosis.     

Calreticulin binding and other biological activities of survival peptide Y-P30 including effects of systemic treatment of rats

Neuron survival-promoting peptide Y-P30, purified from oxidatively stressed neural cell lines, inhibits the appearance of microglia and rescues neurons 1 week after direct application to lesions of the rat cerebral cortex (7). Y-P30 affinity matrices treated with solubilized membranes from a variety of cell lines including human neuroblastoma SY5Y, mouse hippocampal cells HN 33.1, and human promonocytes HL-60, as well as with cerebral cortex tissue from both humans and rats, showed highly specific binding to calreticulin, a ubiquitous calcium binding protein that may be critical for integrin function. Treatment of cultures with 0.1 nM Y-P30 stabilized all these cell types whether differentiated or not, while 1 microM peptide also inhibited the morphological differentiation of the HL-60 cells into macrophages. Western analysis of the medium of SY5Y cell cultures suggested Y-P30-stimulated release of calreticulin, a result consistent with its other biological activities. Likewise, single dose systemic application of Y-P30 in unoperated rats and in rats with cerebral cortex lesions produced significant reductions in cerebral cortex membrane-associated calreticulin. Both direct and intravenous treatment with peptide also reduced cortical neuron atrophy 4 days after these lesions but only direct application consistently inhibited the appearance of ED-1(+) monocyte derivatives. We suggest that in vitro and in vivo mechanisms of Y-P30 effects are similar and involve the targeting of calreticulin. The results also suggest that some of these activities are apparent in the cerebral cortex after systemic application of this peptide.     

GABA-immunoreactive internuclear neurons in the ocular motor system of lampreys

The presence of internuclear neurons in the abducens and oculomotor nuclei of lampreys [González, M.J., Pombal, M.A., Rodicio, M.C. and Anadón, R., Internuclear neurons of the ocular motor system of the larval sea lamprey, J. Comp. Neurol. 401 (1998) 1-15] indicates that coordination of eye movements by internuclear neurons appeared early during the evolution of vertebrates. In order to investigate the possible involvement of inhibitory neurotransmitters in internuclear circuits, the distribution of gamma-aminobutyric acid (GABA) in the extraocular motor nuclei of the lamprey was studied using immunocytochemical techniques. Small GABA-immunoreactive (GABAir) neurons were observed in the three ocular motor nuclei. Numerous GABAir neurons were observed in the group of internuclear neurons of the dorsal rectus oculomotor subnucleus. A second group of GABAir neurons was observed among and below the trochlear motoneurons. Two further groups of GABAir interneurons, periventricular and lateral, were located in the abducens nucleus among the cells of the caudal rectus and the ventral rectus motor subnuclei, respectively. In addition to the presence of GABAir neurons, in all the ocular motor nuclei the motoneurons were contacted by numerous GABAir boutons. Taken together, these results suggest that GABA is involved as a neurotransmitter in internuclear pathways of the ocular motor system of lampreys.     

900MHz微波电磁辐射对原代培养的大鼠脑皮质神经元能量代谢的影响

目的　研究 90 0MHz微波电磁辐射对原代培养的大鼠脑皮质神经元能量代谢的影响。方法　将大鼠脑皮质神经元暴露于 90 0MHz的连续性微波电磁辐射 (SAR =3 2 2mW g、PD =9mW cm2 ) ,每天暴露 2h ,连续 4d或 5d ,及一次性 1 2h暴露 ,以细胞色素氧化酶为观察指标 ,研究微波对神经元能量代谢的影响。结果　微波电磁辐射可使神经元细胞色素氧化酶活性降低。结论　神经元细胞色素氧化酶活性的改变并非“致热效应”所致 ;微波电磁辐射对神经元细胞色素氧化酶活性影响有蓄积毒性作用 ,其影响在一定程度上是可恢复的 ,并且与神经元接受微波辐射时细胞培养年龄关系不密切。     

压力及维生素B1作用下视网膜神经细胞p53、MDM2及Ref1基因的表达

目的:探讨压力及压力加维生素B1联合作用下SD大鼠视网膜神经细胞p53、MDM2及Ref1基因的表达情况。方法:新生SD大鼠视网膜神经细胞体外培养,倒置相差显微镜观察细胞的生长情况,细胞分单纯加压组、实验加压组(加入维生素B1)及未加压对照组,于加压后2 d(培养第9~10天)行苔盼兰染色检查计数细胞成活率,p53免疫组化检查并提取细胞总RNA,用逆转录聚合酶链反应法检测p53、MDM2及Ref1基因的表达。结果:单纯压力组p53／MDM2基因的表达较正常未加压组增强,Ref1基因表达无增强;而维生素B1作用后的加压组,其p53／MDM2基因的表达较单纯加压组明显减弱。结论:压力作用下视网膜神经细胞的损害有凋亡机制参与,维生素B1对压力作用下视网膜神经细胞具有保护作用。     

铅对小鼠海马[Ca2+]的影响及L型钙通道的作用

目的　探讨铅对分离的小鼠海马细胞内游离钙的影响及其及L型钙通道的作用。方法　采用低浓度胰蛋白酶消化法分离小鼠海马细胞 ,并以莹光指标剂 (Fura 2 )作Ca2 荧光探针 ,用双波长荧光法测定海马细胞 [Ca2 ] i。结果　铅可致分离的小鼠海马细胞 [Ca2 ] i 升高 [由静息时的 (2 0 3 4± 10 86)nmol L升至 (4 2 3 3± 19 2 6)nmol L] ,而不论胞外是否有钙 ;铅可抑制钾诱发海马细胞 [Ca2 ] i 升高 ,尼莫地平可加强这种抑制作用 ,而BayK864 4则可部分消除这种抑制作用。结论　铅可致分离的小鼠海马细胞 [Ca2 ] i 升高作用与胞内钙库释放有关 ;铅的抑制钾诱发海马细胞 [Ca2 ] i 升高作用与其抑制L型钙通道有关。