雷公藤内酯对癫痫大鼠神经元PI3K/Akt/mTOR蛋白的影响

目的 通过检测癫痫大鼠海马神经元PI3K、Akt和mTOR蛋白表达,探讨雷公藤内酯抑制癫痫大鼠神经元凋亡的分子机制.方法 30只大鼠随机分为对照组、海人酸组、雷公藤内酯干预组,免疫组化法检测各组大鼠海马神经元PI3K、Akt和mTOR蛋白的表达情况.结果 海人酸组神经元胞体皱缩,形态不规则,数量减少,而雷公藤内酯干预组神经元的数量和形态与对照组相似,海人酸组海马神经元PI3K、Akt、mTOR蛋白表达与对照组比较均减少,而雷公藤内酯干预组海马神经元的PI3K、Akt、mTOR蛋白表达均较海人酸组增加,差异均有统计学意义(P＜0.05).结论 雷公藤内酯可能通过上调PI3K/Akt/mTOR信号通路蛋白表达对癫痫大鼠海马神经元发挥保护作用.     

雷公藤内酯对海人酸致痫大鼠海马神经元的保护作用及相关PUMA蛋白表达的影响

目的探讨雷公藤内酯对海人酸致痫大鼠海马神经元的保护作用及对海马神经元PUMA(P53上调的细胞凋亡调控因子)蛋白表达的影响。方法 30只SD大鼠随机分成3组,其中对照组10只,海人酸组10只,雷公藤内酯干预组10只。结晶紫染色观察海马神经元的形态变化,免疫组化染色法检测海马神经元PUMA蛋白的表达。结果结晶紫染色结果显示,海人酸组中,海马神经元变性或丢失,胞体皱缩,形态不规则。而雷公藤内酯干预组中海马神经元的数量和形态与对照组相似,胞浆清晰淡染,核仁清楚。免疫组化染色结果显示,海人酸组中海马神经元PUMA蛋白表达水平与对照组比较显著增多(P<0.05);雷公藤内酯干预组中海马神经元PUMA蛋白表达水平与海人酸组比较显著减少(P<0.05)。结论雷公藤内酯对海人酸致痫大鼠海马神经元具有保护作用;其可下调海马神经元PUMA蛋白的表达,抑制海人酸致痫大鼠海马神经元凋亡。     

α-细辛醚对氯化锂-匹罗卡品致痫大鼠PI3K/ Akt /mTOR通路的调节作用

目的 探讨α- 细辛醚对癫痫大鼠学习记忆的影响及分子机制。方法 SD 大鼠随机分成 生理盐水组、癫痫组、α-细辛醚组,采用氯化锂-匹罗卡品注射诱导大鼠癫痫造模,α-细辛醚组造模 前用α-细辛醚干预28 d。水迷宫检测大鼠空间学习记忆能力,Western blot方法检测大鼠海马组织PI3K、 Akt、mTOR蛋白表达,采用RT-PCR技术检测大鼠海马组织PI3K、Akt、mTOR 的 mRNA表达。结果 水 迷宫实验结果显示α-细辛醚能够改善大鼠的学习记忆能力;Western blot 结果显示,癫痫组海马组织 PI3K、p-Akt、mTOR 蛋白表达较生理盐水组降低（P＜ 0. 05） ,α-细辛醚组海马组织的各蛋白表达较癫 痫组增加（P＜ 0. 05）。RT-PCR 结果显示,癫痫组海马组织PI3K、p-Akt、mTOR mRNA表达水平与生理盐 水组比较显著降低（P＜ 0. 05）,α-细辛醚组海马组织PI3K、p-Akt 的mRNA表达水平与癫痫组比较显著 增高（P＜ 0. 05） 。结论 α-细辛醚能够改善氯化锂-匹罗卡品致痫大鼠学习记忆,其分子机制可能与 上调PI3K/Akt /mTOR 表达有关。     

雷公藤内酯醇对癫痫大鼠电压门控钾通道kv1.1表达的影响

目的 探讨雷公藤内酯醇(TL)对癫痫大鼠神经的保护作用及其机制.方法 60只SD大鼠分成对照组、模型组、雷公藤组,每组各20只.雷公藤组大鼠腹腔注射TL(每日15μg/kg),模型组大鼠腹腔注射等量生理盐水,注射7d后,雷公藤组与模型组通过颈内皮下注射海人酸(KA)致痫,对照组则颈内皮下注射等量的生理盐水.用免疫组化和Western blot方法检测大鼠海马CA3区瞬时外向钾离子通道kv1.1蛋白表达.结果 模型组大鼠海马CA3区kv1.1蛋白表达水平低于对照组(P＜0.05);雷公藤组海马CA3区kv1.1蛋白表达高于模型组(P＜0.05);雷公藤组与对照组大鼠kv1.1蛋白表达水平无明显差异(P＞0.05).结论 TL对KA致痫大鼠神经元有保护作用,其作用的发挥可能与TL可增加海马CA3区神经元kv1.1的表达有关.     

海人酸癫痫大鼠模型的TNF-α及其mRNA表达特征

目的立体定向手术建立海人酸颞叶癫痫模型,检测癫痫大鼠海马内TNF-α及其mRNA的表达, 评价其意义。方法大鼠一侧海马CA3区注射海人酸,观察其行为学特征及HE染色的病理学改变,免疫组化和原位杂交法检测大鼠海马内TNF—α蛋白和mRNA的动态表达。结果大鼠注射海人酸后出现湿狗样抖动、头面部肌阵挛、肢体阵挛及全面强直阵挛发作等,病理可见海马神经元变性、缺失及胶质细胞增生,海马内TNF-α蛋白与 mRNA表达时程基本一致,3h出现,12h达高峰,而后逐渐下降,7d后回归至对照组表达水平,15d,30d又高于对照组。结论在一侧海马注射海人酸的大鼠癫痫模型中,内源性TNF—α参与了癫痫的发病机制。     

基于PI3K/Akt通路研究右美托咪定对癫痫持续状态大鼠海马神经元自噬的调节作用

目的 探讨右美托咪定(Dexmedetomidine,Dex)对癫痫持续状态(Status epilepticus,SE)大鼠海马神经元自噬的调节作用及对磷脂酰肌醇-3-羟激酶/蛋白激酶B(PI3K/Akt)信号通路的影响。方法 采用戊四氮(PTZ)点燃制备SE大鼠模型,随机分为模型组(M组)、Dex低剂量(Dex-L)组、Dex高剂量(Dex-H)组、Dex-H +LY294002(Dex-H+LY)组,另取正常SD大鼠为对照组(NC组),每组各15只; Dex-L组、Dex-H组分别腹腔注射Dex 25、100 μg/kg; Dex-H+LY组脑室注射5 μL PI3K抑制剂LY294002+腹腔注射Dex 100 μg/kg,NC组、M组大鼠腹腔注射等量生理盐水; 观察大鼠行为学表现并进行脑电图描记; 原位末端标记法(TUNEL)检测各组大鼠海马神经元凋亡情况; 免疫印迹(WB)检测各组大鼠海马组织中LC3、PI3K、p-PI3K、Akt、p-Akt蛋白表达水平。结果 NC组大鼠脑电图无异常放电现象,主要以α、β波为主; M组大鼠出现大量阵发性棘波、高幅尖波、棘慢复合波、尖慢复合波等; Dex-L组、Dex-M组大鼠癫痫发作减少或波幅降低; Dex-H+LY组大鼠癫痫样放电较Dex-H组明显增加。与NC组比较,M组大鼠海马神经元凋亡细胞数、海马LC3-Ⅱ蛋白表达显著增加,海马LC3-Ⅰ,p-PI3K/PI3K,p-Akt/Akt蛋白表达水平显著降低(P<0.05); 与M组比较,Dex-L组、Dex-H组大鼠海马神经元凋亡细胞数、海马LC3-Ⅱ蛋白表达依次减少(P<0.05),海马LC3-Ⅰ,p-PI3K/PI3K,p-Akt/Akt蛋白表达水平依次增高(P<0.05); 与Dex-H组比较,Dex-H+LY组大鼠海马神经元凋亡细胞数、海马LC3-Ⅱ蛋白表达显著增加,海马LC3-Ⅰ,p-PI3K/PI3K,p-Akt/Akt蛋白表达水平显著降低(P<0.05)。结论 Dex可能通过促进PI3K/Akt信号通路激活来抑制SE大鼠海马神经元过度自噬,减轻神经元凋亡,发挥抗惊厥及脑保护作用。     

海人酸致痫大鼠海马NMDAR2B及ERK1/2蛋白表达的变化

目的 通过研究海人酸致痫大鼠海马NMDAR2B及ERK1/2蛋白表达的变化,为进一步探明癫痫脑损伤的机制奠定基础.方法 采用在立体定位仪下,将海人酸注射至大鼠杏仁核的方法制备癫痫模型.将大鼠随机分为正常对照组,假手术组及致痫组,分别于不同时间取材,应用免疫组化的方法观察脑组织NMDAR2B蛋白及ERK1/2蛋白表达的变化.结果 正常海马各区均有极少量的NMDAR2B阳性细胞分布,海人酸致痫组后2h大鼠海马各区NMDAR2B表达迅速增加,6h达高峰,并持续至至癫痫后24h(P＜0.01),ERK1/2蛋白致痫后半小时表达开始增高,3h达高峰,后开始下降,6h后恢复到致<痫前的水平(P＜0.01).结论 NMDAR2B参与了癫痫发生和癫痫脑的长时程的兴奋过程,ERK1/2短时程参与了癫痫的发生过程.     

癫痫动物模型的建立及多药耐药基因产物P-糖蛋白的表达

目的 建立局限性癫痫动物模型,探讨多药耐药基因产物P-糖蛋白的表达规律以及癫痫耐药机制。方法 42只Wistar大鼠随机分为对照组,海人酸致痫组,苯妥英钠(phenytoin sodium,PHT)+苯巴比妥(phenobarbital,PB)和加巴喷丁(gabapentin)抗癫痫药物干预组。致痫组大鼠于右侧海马区注射海人酸,于给药后第6h、24h、3d、5d和7d处死;干预组大鼠在注射海人酸前30min,分别行加巴喷丁(20mg/kg)或PHT(3～90mg/kg)+PB(30mg/kg)腹腔注射,然后注射海人酸,给药后第6h、3d、5d和7d处死。所有动物均采用免疫组织化学法检测多药耐药基因-1的表达产物P-糖蛋白。结果 对照组大鼠无癫痫发作。致痫组大鼠均出现癫痫发作,经抗癫痫药物干预的大鼠,癫痫发作的开始时间延迟、持续时间缩短(P<0.01)。不同剂量PHT组大鼠癫痫发作开始和持续时间有所不同,组间比较差异有显著性意义(P<0.01);随PHT剂量的增加,P-糖蛋白表达增高。干预组阳性细胞数高于致病组(P<0.01)。注射海人酸后6h,出现P-精蛋白强烈表达,3～5d后减弱,7d后完全消失;但干预组大鼠,在给药后第3d仍强烈表达,并逐渐增强,直至第7d仍无减弱趋势。结论 (1)一侧海马区注射海人酸,可成功地建立癫痫模型;(2)抗癫痫药物可使多药耐药基因-1的表达产物P-糖蛋白表达增强,且随药物剂量增加而     

雷公藤内酯通过下调CⅡTA抑制BV-2小胶质细胞MHCⅡ表达

目的观察雷公藤内酯对海人酸活化的BV-2小胶质细胞MHCⅡ分子表达的影响,并探讨其相关的分子机制。方法将BV-2细胞随机分为对照组、海人酸组、雷公藤內酯组,免疫组化方法检测雷公藤内酯对海人酸活化的BV-2细胞MHCⅡ和CⅡTA蛋白的影响。结果对照组MHCⅡ阳性BV-2细胞为(0.059±0.005),海人酸组MHCⅡ阳性BV-2细胞为(0.893±0.038),经统计学处理二者存在显著性差异(P0.05)。雷公藤内酯组MHCⅡ阳性BV-2细胞率(0.089±0.013),与海人酸组比较二者存在显著性差异(P0.05);对照组CⅡTA阳性BV-2细胞为(0.043±0.003),海人酸组CⅡTA阳性BV-2细胞为(0.692±0.015),经统计学处理二者存在显著性差异(P0.05)。雷公藤内酯组CⅡTA阳性BV-2细胞为(0.057±0.009),与海人酸组比较存在显著性差异(P0.05)。结论雷公藤内酯可以抑制海人酸活化的BV-2细胞MHCⅡ表达,其机制可能与下调BV-2细胞CⅡTA表达有关。     

Bcl-2、Bad在大鼠癫痫持续状态中的表达及rHuEpo干预的分子机制

目的观察PTZ致痫的SD大鼠SE时海马神经元损伤的情况,给予rHuEpo干预后的变化以及应用PI3K抑制剂LY294002后神经元凋亡以及Bcl-2、Bad的变化情况,对rHuEpo的作用机制进行更深一步的探讨。方法采用PTZ致痫大鼠SE模型,随机将大鼠分为正常对照组、模型组、rHuEpo干预组、LY294002干预组、LY294002对照组,用TUNEL法检测海马神经元凋亡状况;免疫组化SP法观察p-Akt、Bcl-2、Bad阳性细胞的表达;每组大鼠海马Bcl-2 mRNA、Bad mRNA的表达采用RT-PCR方法检测,每组大鼠海马p-Akt、Bcl-2、Bad蛋白的表达采用Western blot方法检测。结果 rHuEpo干预后可以上调p-Akt、Bcl-2 mRNA和Bcl-2蛋白的表达、减少Bad mRNA和Bad蛋白的表达,对神经元具有一定的保护作用;与rHuEpo干预组相比,PI3K抑制剂LY294002干预后,pAkt、Bcl-2 mRNA和蛋白的表达明显减少,海马Bad mRNA和蛋白的表达较rHuEpo干预组明显增加,rHuEpo的神经保护作用被明显减弱,差异具有统计学意义。结论从正反两个方面提示rHuEpo的抗凋亡作用可能通过对PI3K/Akt信号通路发挥影响,从而对凋亡线粒体途径中Bcl-2、Bad蛋白的表达程度产生调节,最终实现对抗癫痫持续状态凋亡损伤的神经保护作用。     

Environmental factors in the development and progression of late-onset Alzheimer＇s disease

Late-onset Alzheimer＇s disease （LOAD） is an age-related neurodegenerative disorder characterized by gradual loss of synapses and neurons, but its pathogenesis remains to be clarified. Neurons live in an environment constituted by neurons themselves and glial cells. In this review, we propose that the neuronal degeneration in the AD brain is partially caused by diverse environmental factors. We first discuss various environmental stresses and the corresponding responses at different levels. Then we propose some mechanisms underlying the specific pathological changes, in particular, hypothalamic-pituitary adrenal axis dysfunction at the systemic level; cerebrovascular dysfunction, metal toxicity, glial activation, and Aβ toxicity at the intercellular level; and kinase-phosphatase imbalance and epigenetic modification at the intracellular level. Finally, we discuss the possibility of developing new strategies for the prevention and treatment of LOAD from the perspective of environmental stress. We conclude that environmental factors play a significant role in the development of LOAD through multiple pathological mechanisms.     

Edema and neuronal apoptosis in the hippocampus and cortex of elderly rats following transient cerebral ischemia/reperfusion injury

BACKGROUND： Previous studies of cerebral ischemia have used young animals, with an ischemic time greater than 5 minutes （safe time limit）. Despite an increased understanding of neuronal apoptosis, it remains uncertain whether brief cerebral ischemic events of 5 minutes or less damage brain tissue in elderly rodents. OBJECTIVE： To investigate the effects of transient cerebral ischemia （5 minutes）/reperfusion injury on brain cortical and hippocampal edema, aquaporin-4 （AQP-4） expression, and neuronal apoptosis in aged rats, and to compare ischemic sensitivity between cortex and hippocampus. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Institute of Cerebrovascular Disease, Qingdao University Medical School from April 2008 to March 2009. MATERIALS： Rabbit anti-AQP-4 polyclonal antibody, TUNEL kit, and SABC immunohistochemistry kit were purchased from Wuhan Boster Bioengineering, China. METHODS： A total of 160 healthy, male, aged 19-21 months, Wistar rats were randomly assigned to 4 groups： sham-surgery, and ischemia 1-, 3-, and 5-minute groups, with 40 rats in each group. The global cerebral ischemia model was established using the Pusinelli four-vessel occlusion, and the three cerebral ischemia groups were subdivided into reperfusion 12-hour, 1-, 2-, 3-, and 7-day subgroups, with 8 rats in each subgroup. The sham-surgery group was subjected to exposure of the first cervical bilateral alar foramina and bilateral common carotid arteries. MAIN OUTCOME MEASURES： The dry-wet weight assay was used to measure brain water content and histopathology of the cortex and hippocampus was observed following hematoxylin-eosin staining. In addition, cortical and hippocampal AQP-4 expression was detected by streptavidin-biotin complex immunohistochemistry, and neuronal apoptosis was detected by the TUNEL method. RESULTS： There was no significant difference in brain water content or AQP-4 expression in the cortex and hippocampus between ischemia 1- and 3-minute groups and the sham-surgery group or brain water content or AQP-4 expression in the cortex between ischemia 5-minute group and sham-surgery group （P 〉 0.05）. However, brain water content and AQP-4 expression in the hippocampus after 5 minutes of cerebral ischemia were significantly increased compared with the sham-surgery group （P 〈 0.05 or P 〈 0.01）. Several TUNEL-positive cells were observed in the cortex and hippocampus of the sham-surgery group and ischemia 1-minute group, as well as in the cortex of the ischemia 3-minute group. In addition, the number of apoptotic neurons in the hippocampus of ischemia 3-minute group and in the cortex and hippocampus of ischemia 5-minute group was significantly increased （P 〈 0.05 or P 〈 0.01 ）. Neuronal apoptosis was increased after 12 hours of ischemia/reperfusion, and it reached a peak by 2 days （P 〈 0.01）. CONCLUSION： Transient cerebral ischemia （5 minutes） resulted in increased hippocampal edema, AQP-4 expression, and neuronal apoptosis. Moreover, cerebral ischemia had a greater effect on neuronal apoptosis than brain edema or AQP-4 expression, and the hippocampus was more sensitive than the cortex.     

Disrupted structural and functional brain connectomes in mild cognitive impairment and Alzheimer＇s disease

Alzheimer＇s disease （AD） is the most common type of dementia, comprising an estimated 60-80% of all dementia cases. It is clinically characterized by impairments of memory and other cognitive functions. Previous studies have demonstrated that these impairments are associated with abnormal structural and functional connections among brain regions, leading to a disconnection concept of AD. With the advent of a combination of non-invasive neuroimaging （structural magnetic resonance imaging （MRI）, diffusion MRI, and functional MRI） and neurophysiological techniques （electroencephalography and magnetoencephaJography） with graph theoretical analysis, recent studies have shown that patients with AD and mild cognitive impairment （MCI）, the prodromal stage of AD, exhibit disrupted topological organization in large-scale brain networks （i.e., connectomics） and that this disruption is significantly correlated with the decline of cognitive functions. In this review, we summarize the recent progress of brain connectomics in AD and MCI, focusing on the changes in the topological organization of large-scale structural and functional brain networks using graph theoretical approaches. Based on the two different perspectives of information segregation and integration, the literature reviewed here suggests that AD and MCI are associated with disrupted segregation and integration in brain networks. Thus, these connectomics studies open up a new window for understanding the pathophysiological mechanisms of AD and demonstrate the potential to uncover imaging biomarkers for clinical diagnosis and treatment evaluation for this disease.     

墨蝶呤还原酶基因与精神分裂症相关性的研究进展

墨蝶呤还原酶（SPR）催化四氢生物蝶呤（BH4）从头合成途径的最后一步反应。SPR基因遗传缺陷或突变可导致BH。的合成紊乱,影响单胺类神经递质（如多巴胺、5-羟色胺及谷氨酸等）的合成或释放,进而参与包括精神分裂症在内的多种神经精神系统疾病的发生发展过程。此外,SPR基因敲除小鼠表现出持续增强的自主活动等类精神分裂症症状,说明该基因在精神分裂症的发病中扮演重要的角色。进一步研究SPR基因及其单核苷酸多态性的功能,可为阐明精神分裂症的发病机制提供重要的线索,也为新一代抗精神病药物的研制及开发开拓新的视野。现对SPR基因与精神分裂症的相关研究做一综述。     

Total saponins of Panax ginseng effects on proliferation and differentiation of human embryonic neural stem cells and in a Parkinson’s disease mouse model

BACKGROUND： Total saponins of Panax ginseng （TSPG） exhibits neuroprotection against Parkinson＇s disease in the substantia nigra. OBJECTIVE： To investigate the effects of TSPG on human embryonic neural stem cells （NSCs） proliferation and differentiation into dopaminergic neurons using in vitro studies, and to observe NSC differentiation in a mouse model of Parkinson＇s disease, as well as behavioral changes before and after transplantation. DESIGN, TIME AND SETTING： In vitro neural cell biology trial and in vivo randomized, controlled animal trial were performed at the Institute of Basic Medical Sciences, Chongqing Medical University between September 2004 and December 2007. MATERIALS： TSPG （purity 〉 95%） was isolated, extracted, and identified by Chongqing Academy of Chinese Materia Medica. Recombinant human basic fibroblast growth factor （bFGF） and recombinant human epidermal growth factor （EGF） were purchased from PeproTech, USA. A total of 25 C57/BL6J mice, aged 18-20 weeks were included. Twenty were used to establish a Parkinson＇s disease model with i.p. injection of MPTP （1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine） and TSPG alone or combined with interleukin-1 （IL-1）-treated NSCs prior to transplantation into the corpus striatum. The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection, serving as the TSPG prevention group. METHODS： Primary NSCs were isolated, cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs. In vitro experiment： （1） to induce proliferation, NSCs were treated with TSPG, EGF＋bFGF, or TSPG＋EGF＋bFGF, respectively; （2） to induce dopaminergic neuronal differentiation, NSCs were treated with TSPG, IL-1, or TSPG＋IL-1, respectively. MAIN OUTCOME MEASURES： In vitro experiment： the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay. Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation. In vivo experiment： differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining. Behavioral changes were evaluated by spontaneous activity frequency, memory function, and score of paralysis agitans. RESULTS： （1） NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining, as well as neurofilament protein and glial fibrillary acidic protein. （2） TSPG significantly increased NSC proliferation, in particular when combined with EGF and bFGF, which was twice as effective as FGF or bFGF alone. TSPG also induced dopaminergic differentiation in NSCs, in particular when TSPG was added together with IL-1, resulting in an effect five times greater than that of IL-1 alone. （3） At day 30 following transplantation, most NSCs in the TSPG prevention group differentiated into dopaminergic neurons, and the scores of paralysis agitans, spontaneous activity, and memory function were significantly increased compared with TSPG alone or TSPG＋IL-1 groups （P 〈 0.05）. CONCLUSION： TSPG stimulated NSC proliferation, in particular when combined with FGF and bFGF. TSPG significantly induced dopaminergic neuronal differentiation of NSCs, and the effect was greater when combined with IL-1. In addition, TSPG greatly improved behavior in the Parkinson＇s disease mouse model following NSC transplantation. Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1, in terms of behavioral improvements in the Parkinson＇s disease mouse model.     

Caspase 3 siRNA decreases apoptosis in cultureC neuronal cells

BACKGROUND： Lentiviral vectors, a type of retroviral vector, are able to infect cells at all phases of cell cycle. They are able to express exogenous target genes in vivo over long periods of time with limited immunological reaction.
OBJECTIVE： To inhibit neuronal apoptosis by blocking the apoptotic cascade reaction, gene silencing of Caspase 3, and transfection of Caspase 3 short hairpin ribonucleic acid （shRNA） into Neuro 2a cells using a lentiviral vector.
DESIGN： TiME-AND SETTING： An observational, genetic engineering cellular biology experiment was performed in Guangzhou Red Cross Hospital and Guangzhou Institute of Traumatic Surgery between March 2007 and June 2008.
MATERIALS： PLL3.7, PCMV-VSV-G, and PH＇8.9∧PR plasmids were provided by the CBR Institute for Biomedical Research, Harvard Medical School, USA. Staurosporine was purchased from Sigma, USA.
METHODS： Caspase 3 siRNA was synthesized and cloned into the PLL3.7 plasmid. The Caspase 3 shRNA-PLL3.7 Ientivirus was generated in 293T cells using a calcium phosphate transfection kit. After the lentivirus was transfected into Neuro 2a cells, apoptosis was induced in both the transfected and untransfected cells by staurosporine. Cell apoptosis was assessed by flow cvtometrv.
MAIN OUTCOME MEASURES： Caspase 3 mRNA expression was measured by RT-PCR and Caspase protein expression was assessed by Western blot. Cellular apoptosis was determined by flow cytometry using Annevin V-PE/Taad-Cy7. RESULTS： The transfection rate of caspase 3 shRNA was 〉 98% using the lentiviral vector, RT-PCR and Western blot results demonstrated that significantly reduced Caspase 3 mRNA and protein expression in the transfected Neuro 2a. The control group exhibited 38.7% Annexin V/7aad-positive cells, which suggested apoptotic anaphase, while only 5.0% cells in the Caspase 3 gene silencing group entered apoptotic anaphase. CONCKUSION： Caspase 3 shRNA inhibited Caspase 3 expression in Neuro 2a ceils and decreased drug-induced apoptosis of Neuro 2a cells.     

Wnt信号通路与骨髓间充质干细胞增殖及分化的关系

骨髓间充质干细胞（bonemarrow—derived mesenchymal stem cells,BMSCs）是骨髓中不同于造血干细胞的一类细胞,其来源丰富,取材简便,易分离、纯化、培养,在一定的条件下可以迅速体外扩增,具有多向分化潜能,可以通过不同的方法被诱导分化成骨细胞、软骨细胞、肌细胞、神经胶质细胞、神经元细胞等,而且它具有低免疫源性,向病变部位迁移的能力,     

Correlation of hypoxia-inducible factor-1 alpha and erythropoietin protein and mRNA to cerebral ischemic tolerance in a focal ischemia/reperfusion model using the twice suture method

BACKGROUND： Numerous studies have shown that transient ischemic preconditioning induces cerebral ischemic tolerance. However, the underlying mechanisms of endogenous protection following ischemic preconditioning remain unclear. OBJECTIVE： To dynamically measure erythropoietin and hypoxia-inducible factor-1α （HIF-1α） mRNA and protein expression at various times following preconditioning, and to investigate effects of erythropoietin and HIF-1α on cerebral ischemic tolerance in a model of focal ischemia/reperfusion established using the twice suture method. DESIGN, TIME AND SETTING： The randomized, controlled study was performed at the Institute of Anatomy, Medical College, Qingdao University, China from March 2006 to March 2007. MATERIALS： Rabbit anti-rat HIF-1α monoclonal antibody and biotinylated goat anti-rabbit IgG （Boster, China）, rabbit anti-rat erythropoietin monoclonal antibody （Santa Cruz Biotechnology, USA）, and one-step RT-PCR kit （Qiagen, Germany） were used in this study. METHODS： A total of 99 healthy, male, Wistar rats were randomly assigned to three groups： sham surgery （n = 9）, non-ischemic preconditioning （n = 45）, and ischemic preconditioning （n = 45）. In the ischemic preconditioning group, rat models of pre-ischemia-reperfusion-ischemia-reperfusion were established by occluding the left middle cerebral artery using the twice suture method. In the non-ischemic preconditioning group, pre-ischemia was replaced by sham surgery. Subsequently, the ischemic preconditioning and non-ischemic preconditioning groups were equally divided into five subgroups according to time of first reperfusion, including 1-, 3-, 7-, 14-, and 21-day subgroups. The sham surgery group received the sham surgery twice. MAIN OUTCOME MEASURES： HIF-la and erythropoietin protein expression was measured in the cerebral cortex, corpus striatum, and hippocampus of the ischemic hemisphere. HIF-1α and erythropoietin mRNA expression were determined in the frontal and parietal cortex of the ischemic hemisphere. RESULTS： （1） Intergroup comparison： compared with the non-ischemic preconditioning group, HIF-1α protein expression significantly increased in the rat cerebral cortex, corpus striatum, and hippocampus in the ischemic hemisphere at 1,3, and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05 or P 〈 0.01）. Erythropoietin protein expression significantly increased in the cerebral cortex, corpus striatum, and hippocampus, as well as HIF-1α and erythropoietin mRNA expression in the frontal and parietal cortex in the ischemic hemisphere, at 3 and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05）. （2） Temporal expression： HIF-1α protein expression in the rat cerebral cortex, corpus striatum, and hippocampus, as well as HIF-la mRNA expression in the frontal and parietal cortex, in the ischemic hemisphere increased at 3 days, and gradually decreased from 7 days following reperfusion in the ischemic preconditioning group. Temporal erythropoietin protein and mRNA expression was consistent with HIF-1α protein expression. （3） Correlation： erythropoietin mRNA expression positively correlated with HIF-1α mRNA expression （r= 0.737, P 〈 0.01）. CONCLUSION： Ischemic preconditioning induced cerebral ischemic tolerance. Pre-ischemiainduced increase in endogenous HIF-1αexpression, as well as its target gene erythropoietin, participated in the formation of cerebral ischemic tolerance.     

高血压脑出血的显微外科治疗进展

高血压脑出血（Hypertensive intrac-rebral hemorrhage,HICH）是具有高发病率、高病死率、高致残率的急性脑血管疾病,占所有脑卒中患者的10%-20%,早期病死率可高达49.4%。随着人口老龄化,其发病率逐年提高;而外科手术的干预,使其病死率有所下降,但致残率居高不下。如何提高手术疗效和患者生存质量,一直是神经外科医师努力的方向。微侵袭血肿清除术因其手术创伤小,恢复快,是目前国内治疗高血压脑出血的重要手段。