重症肌无力单链抗体融合蛋白的酵母菌表达及特异性鉴定

目的: 真核表达抗人乙酰胆碱受体单链抗体637(scFv637)与人血清白蛋白(HSA)的融合蛋白,以提高融合蛋白的产量和增强其生物学活性.方法: 将携带有重组质粒的毕赤酵母菌GS115用G-418筛选高拷贝重组转化子克隆,诱导表达目的蛋白,表达产物用斑点杂交试验检测,并以SDS-PAGE鉴定融合蛋白的相对分子质量(Mr),应用间接免疫荧光技术确定融合蛋白与人肋间肌乙酰胆碱受体(AChR)的亲和性.结果: 斑点杂交试验显示成功表达出目的蛋白,其Mr约为97 400,在人肋间肌细胞膜之间有荧光出现.结论: 在毕赤酵母菌GS115中成功表达出scFv637-HSA融合蛋白,并且融合蛋白可以与人肋间肌AChR结合,这为进一步研究融合蛋白的功能和应用奠定了基础.     

辅助检查方法对新发病重症肌无力的诊断价值

目的 观察辅助检查方法对尚未经免疫治疗的新发病重症肌无力(MG)患者的诊断价值.方法 回顾性分析156例诊断明确的新发病MG患者初诊时的临床电生理检查[主要包括重复电刺激(RNS)和单肌纤维肌电图(SFEMG)]、新斯的明试验、乙酰胆碱受体抗体(AChRAb)等资料.结果 156例新发病MG患者(无Ⅰ型患者)新斯的明试验以及SFEMG、RNS和AChRAb检查阳性者分别为135(86.5%)、129(82.7%)、108(69.2%)和99(63.5%)例.新斯的明试验和SFEMG阳性检出率差异无统计学意义(P>0.05),但均明显高于RNS和AChRAb阳性检出率(P<0.05).结论 MG的诊断需要进行临床综合判断,各项辅助检查不可相互替代.对新发病MG患者规范化的新斯的明实验和SFEMG检查敏感性高.     

血管性痴呆大鼠海马神经型胆碱能受体的变化

目的 探讨血管性痴呆(VD)大鼠海马神经型胆碱能受体(nAChR)的变化.方法 改良四血管法建立VD大鼠模型;术后1个月,采用Morris水迷宫试验检测VD大鼠的学习和记忆功能;应用改良Ellman法测定大鼠血浆及海马乙酰胆碱酯酶(AChE)活性;Western Blot法及RT-PCR法分别检测大鼠海马nAChR亚单位α3、α4、α7蛋白及mRNA的表达水平,并与假手术组比较.结果 与假手术组相比,VD组水迷宫试验逃避潜伏期明显延长,第1次穿越平台时间延长,穿越平台次数减少(均P<0.01);血浆AChE活性降低(P<0.01),海马AChE活性升高(P<0.05);海马nAChR α3、α4、α7蛋白及nAChR α3、α7 mRNA表达水平明显下降(P<0.05～0.01),nAChR α4 mRNA表达水平明显升高(P<0.01).结论 海马神经元nAChR表达的降低可能是VD大鼠认知功能受损害的基础.     

莲房原花青素对东莨菪碱所致小鼠记忆获得性障碍的改善作用

目的观察莲房原花青素（LSPC）对东莨菪碱所致小鼠学习记忆障碍的影响。方法采用东莨菪碱致小鼠记忆障碍模型,以行为学实验（Morris水迷宫实验、跳台实验）和乙酰胆碱酯酶活性为指标观察LSPC对小鼠记忆障碍的作用。结果Morris水迷宫实验结果显示,LSPC可缩短东莨菪碱所致记忆障碍模型小鼠的潜伏期,减少游泳距离;跳台实验结果显示,LSPC可延长其平台停留期,显著减少错误次数;乙酰胆碱酯酶活性检测结果显示LSPC能显著抑制其乙酰胆碱酯酶活性。结论LSPC对东莨菪碱所致小鼠学习记忆能力障碍有改善作用,其机制之一是基于对胆碱能系统的影响。     

烟碱型乙酰胆碱受体在离体海马小胶质细胞上的表达与定位

目的 探讨烟碱型乙酰胆碱受体(α7-nAChR)在体外培养海马小胶质细胞上的表达和定位.方法 取新生SD大鼠的海马组织,进行胶质细胞的混合培养,然后分离纯化小胶质细胞,采用免疫荧光染色检测CD11b/c进行小胶质细胞的鉴定;采用RT-PCR和免疫荧光双标分别检测α7-nAChR在小胶质细胞中的表达和定位.结果 免疫荧光染色显示分离纯化的细胞表达小胶质细胞特异性抗体CD11b/c;RT-PCR能扩增出长度为450 bp的α7-nAChR目的 条带,免疫荧光双标检测显示小胶质细胞CD11b/c和α7-nAChR染色阳性,分别呈红色和绿色荧光,叠加后呈棕黄色,且细胞膜荧光信号较强.结论 α7-nAChR在体外培养的海马小胶质细胞中能正常表达,定位在细胞膜的功能性α7-nAChR较丰富.     

Effect of Schisandra chinensis polysaccharide on intracerebral acetylcholinesterase and monoamine neurotransmitters in a D-galactose-induced aging brain mouse model

BACKGROUND： The most prominent characteristic of brain aging is decreased learning and memory ability. The functions of learning and memory are closely related to intracerebral acetylcholinesterase （ACHE） and monoamine neurotransmitter activity. Previous studies have shown that Schisandra chinensis polysaccharide has an anti-aging effect. OBJECTIVE： To explore the effects of Schisandra chinensis polysaccharide on AChE activity and monoamine neurotransmitter content, as well as learning and memory ability in a D-galactose-induced aging mouse brain model compared with the positive control drug Kangnaoling. DESIGN, TIME AND SETTING： Completely randomized, controlled experiment based on neurobiochemistry was performed at the Pharmacological Laboratory, Henan University of Traditional Chinese Medicine from September to December 2003. MATERIALS： Schisandra chinensis was purchased from Henan Provincial Medicinal Company. Schisandra chinensis polysaccharide was obtained by water extraction and alcohol precipitation. Kangnaoling pellets were provided by Liaoning Tianlong Pharmaceutical （batch No. 20030804; state drug permit No. H21023095）. A total of 50 six-week-old Kunming mice were randomly divided into five groups： blank control, model, Kangnaoling, high and low dosage Schisandra chinensis polysaccharide groups, with 10 mice per group. METHODS： Mice in the blank control group were subcutaneously injected with 0.5 mL/20 g normal saline into the nape of the neck each day, while the remaining mice were subcutaneously injected with 5% D-galactose saline solution （0.5 mL/20 g） in the nape for 40 days to induce a brain aging model. On day 11, mice in the high and low dosage Schisandra chinensis polysaccharide groups were intragastrically infused with 20 mg/mL and 10 mg/mL Schisandra chinensis polysaccharide solution （0.2 mL/10 g）, respectively. Mice from the Kangnaoling group were intragastrically infused with 35 mg/mL Kangnaoling suspension （0.2 mL/10 g）, and the mice in the model group were intragastrically infused with the same volume of normal saline （0.2 mL/10 g） once per day for 30 consecutive days. MAIN OUTCOME MEASURES： Two hours after the final administration, pathohistological changes in the cerebral cortex and hippocampus were observed using hematoxylin ＆ eosin staining. AChE activity was detected using chromatometry. Monoamine neurotransmitter content was measured using fluorimetry. Learning and memory was measured using the step down test and darkness avoidance test. RESULTS： Both Schisandra chinensis polysaccharide and Kangnaoling improved pathological injury to the cerebral cortex and hippocampus in a mouse model of brain aging. Compared with the blank control group, AChE activity and content of norepinephrine （NA）, dopamine （DA）, and 5-hydroxytryptamine （5-HT） were significantly decreased in the model group （P 〈 0.01 ）. In contrast, AChE activity and NA, DA, and 5-HT levels significantly increased in the Kangnaoling and high dosage Schisandra chinensis polysaccharide groups （P 〈 0.01）, while NA levels significantly increased in the low dosage Schisandra chinensis polysaccharide group （P 〈 0.01）. Drug treatment improved learning and memory abilities （P 〈 0.01 or P 〈 0.05）. CONCLUSION： Schisandra chinensis polysaccharide significantly increased levels of central neurotransmitters and improved learning and memory in a mouse model of brain aging. The effects of Schisandra chinensis polysaccharide were equal to that of Kangnaoling pellets.     

4002/大鼠海马中83KD蛋白诱导神经干细胞向AChE阳性神经元定向分化的作用（英文）

背景：神经干细胞的临床应用还尚待时日,现阶段需要解决如何诱导神经干细胞分化为特定表型的神经元以替代丢失、变性的神经元细胞。 目的：探讨大鼠海马组织83 ku蛋白对神经干细胞向乙酰胆碱酯酶阳性神经元分化的作用。 设计、时间及地点：细胞学体外对照观察,于2003-10/2008-04在南通大学医学院完成。 材料：清洁级SD大鼠12只,17 d龄SD胎鼠多只,均由南通大学实验动物中心提供。 方法：取6只正常大鼠及6只切割海马伞后14 d大鼠的海马组织制成匀浆,进行非变性聚丙烯酰胺凝胶电泳,根据染色结果切取含有83 ku差异蛋白条带进行电洗脱,定量后调整蛋白浓度为300 mg/L。取胎鼠前脑组织,体外分离培养神经干细胞,设立3组：空白对照组加入单纯DMEM/F12无血清培养基;83 ku蛋白正常组、83 ku蛋白切割组分别加入含10 mg/L来自正常/割海马伞大鼠海马组织83 ku蛋白的DMEM/F12无血清培养基,诱导12 d。 主要观察指标：用乙酰胆碱酯酶组织化学染色检测神经干细胞分化为乙酰胆碱酯酶阳性神经元的情况。 结果：诱导12 d后,83 ku蛋白切割组乙酰胆碱酯酶阳性神经元较多,胞体大且分化较好,突起粗且长;83 ku蛋白正常组乙酰胆碱酯酶阳性神经元较少,胞体小,突起短;空白对照组仅见少量乙酰胆碱酯酶阳性神经元。组间乙酰胆碱酯酶阳性神经元数比较差异有显著性意义(P < 0.05),83 ku蛋白切割组>83 ku蛋白正常组>空白对照组。 结论：大鼠海马组织中83 ku蛋白可成功诱导神经干细胞定向分化为乙酰胆碱酯酶阳性神经元。     

Axonal regeneration from the spinal cord to peripheral nerve induced by end-to-side neurorrhaphy Evidence from acetylcholinesterase staining and Fluorogold retrograde tracing

BACKGROUND： In recent years, surgeons have advocated root or trunk repair of avulsed nerve roots for overall recovery. However, donor nerves pose a major problem, because they do not contain adequate numbers of axons. Moreover, the procedures lead to nerve deficits in the donor nerve following transplantation. OBJECTIVE： To observe whether axonal regeneration occurs by end-to-side neurorrhaphy in the peripheral nerve and spinal cord. DESIGN, TIME AND SETTING： A neuroanatomical, randomized, controlled, animal study was performed at Functional Anatomy Lab in Nagoya University School of Medicine from May 2002 to July 2003. MATERIALS： Fluorogold was purchased from Fluorochrome, LLC, USA. BX50 light microscope and fluorescent microscope were purchased from Olympus, Japan. METHODS： A total of 21 rats were randomly divided into three groups, and the posterior avulsion injury model （C6-8） of the brachial plexus was performed. In the ventral root graft group, the avulsed C7 ventral roots were reanastomosed to the small anterior lateral aspect window of the spinal cord via nerve grafts. In the dorsal root graft group, the C7 dorsal roots were reanastomosed at the small pia mater window of the posterior lateral aspect of the spinal cord via nerve grafts. In the control group, the avulsed nerve roots were not repaired. MAIN OUTCOME MEASURES： The nerve grafts were collected from the ventral and dorsal root graft groups, and the C7 proximal nerve end was collected from the control group. Acetylcholinesterase staining was performed on the tissue. Fluorogold retrograde tracing technique was applied to determine the origin of the regenerating axons. RESULTS： Results showed that acetylcholine-positive axons existed in nerve grafts of the ventral and dorsal root graft groups. However, axons were not found in the avulsed nerve roots of the control group. Fluorogold retrograde tracing confirmed the presence of fluorogold-containing neurons in the ventral and dorsal horn of the ventral and dorsal root graft groups. Fluorogold-positive neurons were not observed in the control group. CONCLUSION： End-to-side neurorrhaphy induced axonal regeneration from the spinal cord to the peripheral nervous system.     

生活

常穿高跟鞋或可导致食欲下降 日本一项研究指出,年轻女性喜欢穿的高跟鞋或瘦型鞋,容易使脚趾受到挤压,血液循环不畅,严重时会影响脑部血液循环,导致摄食中枢的下丘脑外侧区域供血不足,从而引起厌食.长时间穿这样的鞋还会引起脚垫和脚底疼痛,导致人体内神经递质乙酰胆碱分泌大大减少,导致食欲下降.     

α-芋螺毒素LvIA特定氨基酸的突变对其活性的影响

α-芋螺毒素(α-Conotoxin, α-CTx)作为烟碱型乙酰胆碱受体(nicotinic acetylcholine receptor, nAChR)的专一性拮抗剂,是研究nAChRs相关药理和生理功能的重要工具。目的 对特异区分α6/α3β2β3和α3β2两种nAChRs亚型的α-CTx LvIA进行研究,确定影响其活性的关键氨基酸位点。 方法 通过序列比对,两步法氧化折叠、双电极电压钳和圆二色谱等技术确定LvIA中影响其活性的关键氨基酸位点,并探索该位点对LvIA影响的机制。结果 确定了LvIA中第五位组氨酸(His,H)的关键作用,当第五位的His分别被丙氨酸(Ala,A)、天冬氨酸(Asp,D)和色氨酸(Trp,W)分别取代后,都会导致LvIA对所有受体亚型的活性丧失。圆二色结果表明该位点的变化对LvIA的二级结构没有显著的影响。结论 第5位His是维持LvIA活性的关键氨基酸残基,该氨基酸极性和侧链的变化均会导致LvIA活性的丧失,但是对其二级结构影响较小。LvIA中第5位氨基酸与nAChR的相互作用力是保证其活性的关键。该结论为基于LvIA设计新型专一作用于乙酰胆碱受体的工具探针改造提供了重要信息。