嘌呤霉素敏感的氨肽酶对β-淀粉样蛋白诱导的细胞凋亡的影响

目的 探讨嘌呤霉素敏感的氨肽酶(PSA)对β-淀粉样蛋白(Aβ25-35)诱导的SH-SY5Y细胞、PC12细胞凋亡的影响. 方法 采用MTT法检测Aβ25-35对SH-SY5Y、PC12细胞生长的影响;Hoechst染色检测Aβ25-35对SH-SY5Y、PC12细胞核形态的影响;进一步采用脂质体转染法在SH-SY5Y细胞中瞬时转染PSA-siRNA,在PC12细胞中瞬时转染PSA重组质粒,加入Aβ25-35作用24h后收集细胞,进行流式细胞术检测细胞凋亡率;Western blotting检测PSA、caspase-3蛋白的表达变化;酶标仪检测caspase-3活性. 结果 Aβ25-35抑制SH-SY5Y、PC12细胞增殖,细胞核发生凋亡的形态学改变,流式检测细胞凋亡率增加;在SH-SY5Y细胞中,沉默PSA表达能使Aβ25-35诱导的细胞凋亡率升高,促进caspase-3酶原激活,caspase-3活性升高.反之,PC12细胞中过表达的PSA能使Aβ25-35诱导的细胞凋亡率下降,抑制caspase-3酶原激活,caspase-3活性降低. 结论 Aβ25-35能抑制神经细胞生长,引起神经细胞凋亡;PSA能抑制Aβ25-35诱导的神经细胞凋亡,对神经元具有保护作用,其机制可能与抑制caspase-3通路的激活有关.     

醒脑静对Aβ诱导的SH-SY5Y细胞凋亡的保护作用及机制

目的探讨醒脑静对Aβ25~35诱导的人神经母细胞瘤SH-SY5Y细胞凋亡的保护作用及其机制。方法建立Aβ25~35诱导的SH-SY5Y细胞凋亡模型;MTT法检测不同浓度醒脑静对Aβ25~35诱导的SH-SY5Y细胞增殖的影响;Annexin-V/PI流式细胞分析法检测不同浓度醒脑静对Aβ25~35诱导的SH-SY5Y细胞凋亡的影响;Western blot法检测细胞中线粒体凋亡相关蛋白Bcl-2及Bax表达水平变化情况。结果 SH-SY5Y细胞经过Aβ25~35造模处理后,细胞形态由梭形变为方形,细胞大小固缩,数量减少,凋亡小体形成,凋亡模型构建成功;醒脑静可以促进Aβ诱导的SH-SY5Y细胞增殖;醒脑静可以抑制Aβ25~35诱导的SH-SY5Y细胞凋亡、下调Bax蛋白表达及上调Bcl-2蛋白表达抑制Aβ25~35诱导的SH-SY5Y细胞凋亡。结论醒脑静对Aβ25~35诱导的SHSY5Y细胞凋亡具有一定的保护作用,其机制可能是通过调节Bcl-2及Bax水平抑制细胞凋亡的发生。     

丁基苯酞对帕金森病细胞模型保护作用的初步研究

目的探讨丁基苯酞(dl-3-n-Butylphthalide,NBP)对鱼藤酮诱导的帕金森病细胞模型的保护作用及其机制。方法分别使用终浓度为0.1、1、10、100μM NBP和溶剂二甲基亚砜(DMSO)预处理SH-SY5Y细胞24h后,加入终浓度为200nM的鱼藤酮处理24h建立多巴胺能细胞损伤模型,观察各组细胞形态,采用四甲基偶氮唑盐(MTT)比色法检测细胞活性,流式细胞术检测细胞凋亡率(Annexin V-FITC/PI)、线粒体膜电位(JC-1)、细胞内活性氧水平(DCFH-DA)。结果200nmol/L鱼藤酮处理SH-SY5Y细胞24h能够诱导细胞活性下降和细胞凋亡,NBP预处理后SH-SY5Y细胞存活率明显升高,细胞凋亡率降低,线粒体膜电位显著升高(P0.05),细胞内活性氧水平显著降低(P0.05),且随NBP浓度的增加对SH-SY5Y细胞的保护作用增强。结论NBP对鱼藤酮诱导的SH-SY5Y细胞损伤具有良好的保护作用,线粒体保护可能是其作用机制之一。     

锂剂对SH-SY5Y细胞氧糖剥夺模型的保护作用及其对AIF凋亡通路的影响

目的 研究不同浓度的锂剂对SH-SY5Y细胞氧糖剥夺模型的保护效果,确定有效的浓度,并探讨其对AIF凋亡通路的作用,分析其神经保护作用的机制.方法 以不同浓度的锂剂对制备氧糖剥夺模型的SH-SY5Y细胞进行预处理,观察其对细胞的保护作用.MTT法描绘细胞生长曲线.Annexin V和PI双染,流式细胞仪检测各组细胞凋亡情况.免疫荧光检测凋亡诱导因子(AIF)蛋白的变化.结果 MTT显示不同浓度的锂剂对SH-SY5Y细胞氧糖剥夺模型有较强的细胞保护作用,1mmol/L氯化锂为细胞保护的有效剂量.Annexin V和PI双染,流式细胞仪检测可见锂剂处理组凋亡细胞明显减少.免疫荧光染色可见锂剂处理组AIF蛋白发生核移位减少.结论 锂剂对氧糖剥夺模型的SH-SY5Y细胞具有较强的保护作用,1mmol/L氯化锂为细胞保护的有效剂量,其作用机制之一可能是对AIF凋亡通路的抑制.     

鱼藤酮建立帕金森病细胞模型的方法研究

目的探讨鱼藤酮诱导SH-SY5Y细胞建立帕金森病(Parkinson disease,PD)细胞模型的方法。方法实验分为空白对照组及鱼藤酮0.5μmol/L、1.0μmol/L、2.5μmol/L、5.0μmol/L组共5组,各组分别于鱼藤酮加入24h、48h点,观察SH-SY5Y细胞形态变化,采用MTT比色法检测细胞活力;于24h时采用Western blot法检测细胞α-突触核蛋白(α-synuclein)表达量;对空白对照组及鱼藤酮0.5μmol/L、1.0μmol/L组于24h、48h行HE染色观察细胞内包涵体形成情况。以激光共聚焦免疫荧光法观察鱼藤酮1.0μmol/L组24h时α-synuclein的分布情况。结果空白对照组细胞生长较密,神经突起细长;在鱼藤酮药物组24时,0.5μmol/L组细胞突起变短,1.0μmol/L组细胞突起短缩更明显,2.5μmol/L组胞体肿胀明显,5.0μmol/L组圆缩细胞增多;各浓度组48h时与24h时比较,细胞突起变短、细胞肿胀、圆缩更明显。MTT试验显示1.0μmol/L鱼藤酮24h组和0.5μmol/L鱼藤酮48h组分别使细胞活性下降到空白对照组的(86.06±9.06)%和(62.06±7.13)%,与对照组比较差异均具有统计学意义(P0.05)。Western blot结果显示鱼藤酮0.5、1.0、2.5和5.0μmol/L组SH-SY5Y细胞内α-synuclein相对表达水平分别为(1.23±0.11)、(1.41±0.20)、(1.77±0.09)和(1.31±0.28),其中1.0μmol/L、2.5μmol/L鱼藤酮组α-synuclein水平高于空白对照组(P0.05);HE染色显示,在1.0μmol/L鱼藤酮24h组和0.5μmol/L鱼藤酮48h组均观察到细胞质内出现嗜伊红、边界清楚的类路易小体样包涵体;免疫荧光染色显示在1.0μmol/L鱼藤酮24h组观察到细胞内出现颗粒状α-synuclein免疫染色阳性聚集体,可被Thioflavin S共定位。结论 1.0μmol/L鱼藤酮24h诱导SH-SY5Y细胞所建立的PD模型能够很好地模拟PD病理改变,可作为研究PD发病机制的细胞模型。     

99mTc-Annexin V检测早期凋亡多巴胺能神经元的实验研究

目的 研究放射性核素标记的膜联蛋白V(Annexin V)与凋亡的多巴胺能神经元的结合特性,探讨使用凋亡显像剂99mTc-Annexin V早期活体显像诊断帕金森病的可行性.方法 采用不同浓度的1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium, MPP+)处理大鼠肾上腺嗜铬细胞瘤细胞(PC12)和人神经母细胞瘤细胞(SH-SY5Y)以诱导其凋亡(PC12 0～200 μm/L, SH-SY5Y 0～500 μm/L),FITC -Annexin V及碘化吡啶(propidiumiodide, PI)双染进行流式细胞仪凋亡检测.以99mTc-Annexin V与凋亡的细胞进行饱和结合实验及细胞摄取实验,研究凋亡细胞与Annexin V的亲和力及其摄取99mTc-Annexin V的动力学.结果 MPP+可诱导PC12细胞及SH-SY5Y细胞发生凋亡,并有明显的量效关系.凋亡细胞可特异性与99mTc-Annexin V结合,亲和力可达(7.16±1.78)nmol/L,每个凋亡的多巴胺能神经元表面的结合位点可达到(179±33)fmol/106cells (PC12)及(220±26)fmol/106cells (SH-SY5Y),且神经元的凋亡水平与其膜结合的99mTc-Annexin V放射性强度有相关性(P＜0.001).结论 凋亡的多巴胺能神经元与99mTc-Annexin V有高度亲和力,其所结合的99mTc-Annexin V放射性强度与细胞的凋亡水平相关,99mTc-Annexin V可用于检测多巴胺能神经元的早期凋亡.     

β淀粉样蛋白25-35致神经细胞损伤的机制及信号转导通路初步研究

目的探讨β淀粉样蛋白25-35(Aβ25-35)导致神经细胞损伤的机制及在损伤过程中涉及的信号转导通路中相关酶caspase-3的变化。方法采用荧光染色法、流式细胞法、反转录PCR法研究Aβ25-35对SH-SY5Y细胞的损伤机制及信号转导通路中caspase-3的变化。结果经Aβ25-35处理的SH-SY5Y细胞核中出现浓染的碎块状荧光,流式细胞法检测到凋亡特有的亚二倍体峰,对照组细胞凋亡率为(3.57±0.28)%(n=9),经10、20μmol/L Aβ25-35处理30 h的细胞,其凋亡率分别为(17.44±1.27)%和(38.82±2.06)%,与对照组比较差异有统计学意义(均P<0.01)。反转录PCR产物凝胶电泳的caspase-3与看家基因-βactin的比(0.92)高于对照组(0.19)。结论Aβ25-35促使SH-SY5Y细胞凋亡,此过程中caspase-3转录水平增高。     

胰岛素样生长因子Ⅰ抑制氧化型低密度脂蛋白诱导内皮细胞凋亡的体外试验*☆

目的：胰岛素样生长因子Ⅰ作为重要的促细胞生长因子之一，其对内皮细胞凋亡影响的报道不多。实验拟验证和探讨胰岛素样生长因子Ⅰ对氧化型低密度脂蛋白诱导人脐静脉内皮细胞凋亡的抑制作用及可能机制。 方法：实验于2006-12/2007-07在华中科技大学同济医学院协和医院心血管疾病研究所完成。①实验材料及分组处理：取人新鲜脐带（产妇或家属签署知情同意书）分离培养人脐静脉内皮细胞,分为: 胰岛素样生长因子Ⅰ组(1×10-9 mmol/L)、氧化型低密度脂蛋白(200 mg/L)+胰岛素样生长因子Ⅰ组(1×10-9 mmol/L)、氧化型低密度脂蛋白(200 mg/L)组、正常对照组。分别在细胞培养24 h后加入。②实验评估：采用四甲基偶氮唑盐法测定细胞活力；DAPI细胞核荧光染色法观察细胞凋亡的形态学变化及细胞凋亡率的测定；并进行内皮细胞 caspase-3活性的检测。 结果：①氧化型低密度脂蛋白可明显抑制人脐静脉内皮细胞增殖，胰岛素样生长因子Ⅰ与氧化型低密度脂蛋白共同加入后，细胞增殖率明显上升（P < 0.05）。②氧化型低密度脂蛋白可诱导人脐静脉内皮细胞发生凋亡，而加入胰岛素样生长因子Ⅰ刺激后细胞凋亡率降低（P < 0.05）。③caspase-3活性检测表明与对照组相比，氧化型低密度脂蛋白能明显上调caspase-3的表达，而胰岛素样生长因子Ⅰ+氧化型低密度脂蛋白组caspase-3活性则降低（P < 0.05）。 结论：胰岛素样生长因子Ⅰ对氧化型低密度脂蛋白诱导的人脐静脉内皮细胞凋亡具有抑制作用，可能与其下调caspase-3蛋白表达有关。     

过表达Mfn2基因通过PI3K/AKT通路抑制鱼藤酮诱导的帕金森病细胞模型的细胞凋亡

目的 探讨过表达线粒体融合蛋白2(Mfn2)基因通过PI3K/AKT通路抑制鱼藤酮诱导的帕金森病细胞模型的细胞凋亡。方法 培养SH-SY5Y细胞并分组,对照组用不含药物及质粒的DMEM干预; 鱼藤酮组用含有20 μmol/L鱼藤酮的DMEM干预; 鱼藤酮+空白质粒组用含有20 μmol/L鱼藤酮的DMEM干预并转染空白的pcDNA 3.1质粒; 鱼藤酮+Mfn2质粒组用含有20 μmol/L鱼藤酮的DMEM干预并转染表达Mfn2的pcDNA3.1质粒; 鱼藤酮+Mfn2质粒+LY组用含有20 μmol/L鱼藤酮、10 μmol/L LY294002的DMEM干预并转染表达Mfn2的pcDNA3.1质粒; 检测细胞活力、凋亡率及线粒体凋亡基因、p-PI3K、p-AKT的表达水平。结果 鱼藤酮组的细胞活力及细胞中p-PI3K、p-AKT的表达水平均低于对照组,凋亡率及细胞中caspase-9、cleaved caspase-3的表达水平均高于对照组; 鱼藤酮+Mfn2质粒组的细胞活力及细胞中p-PI3K、p-AKT的表达水平均高于鱼藤酮组,凋亡率及细胞中caspase-9、cleaved caspase-3的表达水平均低于鱼藤酮组; 鱼藤酮+Mfn2质粒+LY组的细胞活力及细胞中p-PI3K、p-AKT的表达水平均低于鱼藤酮+Mfn2质粒组,凋亡率及细胞中caspase-9、cleaved caspase-3的表达水平均高于鱼藤酮+Mfn2质粒组。结论 过表达Mfn2基因对鱼藤酮诱导的帕金森病细胞模型的细胞凋亡具有抑制作用,且该作用可能与抑制PI3K/AKT通路有关     

Denervation study of synapses of organ of corti of old world monkeys

Fine structural characteristics of synapses in the spiral organ of Corti were examined, with reference to differences between inner and outer haircell systems, and to location of neurons of origin of efferent axons. Surgical interruption of crossed olivocochlear bundle, of vestibular nerve, of facial nerve, and excision of superior cervical ganglia were used to determine the pathways of efferent axons. Interruption of the vestibular nerve near the brainstem results in degeneration of all efferent terminals on outer hair cells. Mid-line lesions at, and caudal to, the facial colliculus result in degeneration of about half of these efferent terminals. Efferent synaptic bulbs to the inner hair-cell system are small, of the order of one micron, and form type 2 junctions with afferent dendrites. They tend to have more large dense-core vesicles (about 80 nm) than the large efferent terminals of the outer hair-cell system, and appear to be the terminals of axons in the habenula perforata, which exhibit varicosities laden with large dense core vesicles. The varicosities are unaffected by excision of the superior cervical ganglia. So far as our material can reveal, it appears that the varicosities in the habenula perforata do not survive vestibular root interruption, nor do the efferent processes in the internal spiral bundle or at the base of inner hair cells. Most interestingly, the afferent processes of the inner hair-cell system, as identified for example by their relation to pre-synaptic bodies in the inner hair cells, are subject to a trans-synaptic reaction after severance of the vestibular root. They undergo a dramatic cytological transformation, characterized by increase of volume, engorgement with microtubules, microfilaments, microvesicles of various sizes, and clusters of lysosomes. Thus, both the efferent and afferent terminals of the inner hair-cell system show marked cytological differences from the corresponding terminals of the outer hair cell system.     

Summary of legislation of 1935 of interest to the department of Mental Hygiene

Psychiatric Quarterly -     

Summary of legislation of 1941 of interest to the Department of Mental Hygiene

Psychiatric Quarterly -     

Asymmetry of on- and off-pathways of blue-sensitive cones of the retina of macaques

Macaque retinal ganglion cells whose receptive-field center recieves input from blue-sensitive cones show an overt asymmetry of the frequency of ON-center and OFF-center varieties, an asymmetry not present in ganglion cells whose center receives input from the other two cone types. A similar asymmetry of ON/OFF responses is found in the local electrotetinogram (d-wave) mediated by signals from blue-sensitive cones. ‘Blue-ON-center’ ganglion cells have larger receptive-field centers and shorter conduction latencies than other opponent-color varieties, suggesting an appreciable degree of receptor convergence and presumably large cell bodies. Intracellular stainings of these neurons with Procion Yellow show that they correspond to diffuse stratified (Parasol) ganglion cells whose flat-topped dendritic arborization stratifies in the sclerad half of the inner plexiform layer. In view of the known characteristics of macaque bipolar cells and of the ON/OFF asymmetry, it is proposed that these ganglion cells are postsynaptic to cone-specific flat bipolars possibly mediating sign-inverting synaptic contacts. The results also indicate a reversal, for the blue-cone pathway, of the ON/OFF lamination of the inner plexiform layer that has recently been described in other species.     

Mechanism of action of tubocurarine on nicotinic cholinoreceptors of neurons of the sympathetic ganglia of rats

Tubocurarine (Tc) effect on membrane currents elicited by acetylcholine (ACh) was studied in isolated superior cervical ganglion neurons of rat using patch-clamp method in the whole-cell recording mode. The "use-dependent" block of ACh current by Tc was revealed in the experiments with ACh applications, indicating that Tc blocked the channels opened by ACh. Mean lifetime of Tc-open channel complex, tau, was found to be 9.8 +/- 0.5 s (n = 7) at -50 mV and 20-24 degrees C. tau exponentially increased with membrane hyperpolarization (e-fold change in tau corresponded to the membrane potential shift by 61 mV). Inhibition of the ACh-induced current by Tc (3-30 microM/1) was completely abolished by membrane depolarization to the level of 80-100 mV. Inhibition of ACh-induced current was augmented at increased ACh doses. It is concluded that the open channel block produced by Tc is likely to be the only mechanism for Tc action on nicotinic acetylcholine receptors in superior cervical ganglion neurons of rat.