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中国临床康复 2005年第22期9卷 80-81,i003页

作者：刘宏志杨永利李佐文铁法煤业集团总医院骨科辽宁省调兵山市112700

目的:探讨脊髓源性神经干细胞向神经元诱导分化过程中生长相关蛋白43的表达,分析其对脊髓损伤修复的主导作用。方法:实验于2003-03/09在中国医科大学实验动物中心完成。取15只孕14d的胎鼠,自胎鼠的脊髓区提取神经干细胞,经培养及神经巢... 详细信息

目的:探讨脊髓源性神经干细胞向神经元诱导分化过程中生长相关蛋白43的表达,分析其对脊髓损伤修复的主导作用。方法:实验于2003-03/09在中国医科大学实验动物中心完成。取15只孕14d的胎鼠,自胎鼠的脊髓区提取神经干细胞,经培养及神经巢蛋白免疫细胞化学鉴定,观察在神经干细胞定向诱导分化为神经元过程中,生长相关蛋白43于不同时间点的形态学表达。培养液为添加B27(2%)、碱性成纤维细胞生长因子(20mg/L)、表皮细胞生长因子(20mg/L)的无血清培养基DMEM/F12。置于37℃、体积分数为0.05的CO2培养箱中悬浮培养,观察其生长情况,每3天换液1次,共培养5次。分别于分化1,2,4,8d取出载玻片,对生长相关蛋白的表达进行免疫组化检测。总细胞和阳性细胞在显微镜下计数。结果:15只胎鼠的脊髓源性神经干细胞全部进入结果分析。①神经干细胞的培养情况:培养24h后,可见神经球;第2天时,神经球数目增多,呈桑椹状,折光性强,死细胞无光泽,仅有轮廓。②神经干细胞的鉴定结果:免疫细胞化学染色可见神经球内圆形细胞呈神经巢蛋白抗原强阳性,核未着色。神经干细胞定向分化后,细胞逐渐贴壁,到第4,8天时,细胞逐渐变大,突起变长,偶见多个突起。神经元特异性烯醇化酶阳性的神经元,胞浆及核膜出现棕色阳性反应,细胞核大而圆,无着色。细胞连续计数共712个,其中神经元特异性烯醇化酶阳性细胞296个,比例为41.57%。③神经干细胞分化过程中生长相关蛋白表达的测定:神经干细胞分化第1天,胞质内出现生长相关蛋白,第4天表达增强,于核周及突起中明显,逐渐向胞膜转移,并且长的突起与其他细胞的突起之间形成连接,至分化第8天时这种突起生长停止,神经干细胞分化为成熟的神经元细胞,生长相关蛋白反应减弱并消失。细胞连续计数共736个,其中生长相关蛋白阳性细胞246个,比例为33.42%。结论:在神经干细胞分化为神经元的过程中,生长相关蛋白基因呈一过性表达,参与引导突起的生长并发挥促进作用。

关键词：干细胞细胞分化神经组织蛋白质类/分析神经系统/细胞学

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Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin

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NEURON 1999年第3期23卷 569-582页

作者： Naisbitt, S Kim, E Tu, JC Xiao, B Sala, C Valtschanoff, J Weinberg, RJ Worley, PF Sheng, M Massachusetts Gen Hosp Dept Neurobiol Howard Hughes Med Inst Boston MA 02114 USA Harvard Med Sch Boston MA 02114 USA Johns Hopkins Univ Sch Med Dept Neurosci Baltimore MD 21205 USA Univ N Carolina Dept Cell Biol & Anat Chapel Hill NC 27599 USA Pusan Natl Univ Dept Pharmacol Pusan 609735 South Korea

NMDA receptors are linked to intracellular cytoskeletal and signaling molecules via the PSD-95 protein complex. We report a novel family of postsynaptic density (PSD) proteins, termed Shank, that binds via its PDZ domain to the C terminus of PSD-95-associated protein GKAP. A ternary complex of Shank/GKAP/PSD-95 assembles in heterologous cells and can be coimmunoprecipitated from rat brain. Synaptic localization of Shank in neurons is inhibited by a GKAP splice variant that lacks the Shank-binding C terminus. In addition to its PDZ domain, Shank contains a proline-rich region that binds to cortactin and a SAM domain that mediates multimerization. Shank may function as a scaffold protein in the PSD, potentially cross-linking NMDA receptor/PSD-95 complexes and coupling them to regulators of the actin cytoskeleton.

关键词：肌动蛋白类/代谢衔接蛋白质类信号转导 COS细胞载体蛋白质类/分析载体蛋白质类/化学载体蛋白质类/遗传学载体蛋白质类/代谢皮动蛋白细胞骨架/化学细胞骨架/超微结构海马/细胞学细胞内信号肽和蛋白质类膜蛋白质类微丝蛋白质类/分析微丝蛋白质类/代谢显微镜检查免疫电子分子序列数据多基因族神经组织蛋白质类/分析神经组织蛋白质类/代谢神经元/化学神经元/细胞学神经元/代谢蛋白质结构三级受体 N-甲基-D-天冬氨酸/分析受体 N-甲基-D-天冬氨酸/代谢序列同源性氨基酸突触/化学突触/代谢突触/超微结构动物兔大鼠

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Role of cytochrome c as a stimulator of α-synuclein aggregation in Lewy body disease

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第41期274卷 28849-28852页

作者： Hashimoto, M Takeda, A Hsu, LJ Takenouchi, T Masliah, E Univ Calif San Diego Sch Med Dept Neurosci La Jolla CA 92093 USA Univ Calif San Diego Sch Med Dept Pathol La Jolla CA 92093 USA Yokohama City Univ Sch Med Dept Psychiat Kanazawa Ku Yokohama Kanagawa 236 Japan

alpha-Synuclein is a major component of aggregates forming amyloid-like fibrils in diseases with Lewy bodies and other neurodegenerative disorders, yet the mechanism by which alpha-synuclein is intracellularly aggregated during neurodegeneration is poorly understood. Recent studies suggest that oxidative stress reactions might contribute to abnormal aggregation of this molecule. In this context, the main objective of the present study was to determine the potential role of the heme protein cytochrome c in alpha-synuclein aggregation. When recombinant alpha-synuclein was coincubated with cytochrome c/hydrogen peroxide, alpha-synuclein was concomitantly induced to be aggregated. This process was blocked by antioxidant agents such as N-acetyl-L-cysteine. Hemin/hydrogen peroxide similarly induced aggregation of alpha-synuclein, and both cytochrome c/hydrogen peroxide- and hemin/hydrogen peroxide-induced aggregation of alpha-synuclein was partially inhibited by treatment with iron chelator deferoxisamine. This indicates that iron-catalyzed oxidative reaction mediated by cytochrome c/hydrogen peroxide might be critically involved in promoting alpha-synuclein aggregation. Furthermore, double labeling studies for cytochrome c/alpha-synuclein showed that they were colocalized in Lewy bodies of patients with Parkinson's disease. Taken together, these results suggest that cytochrome c, a well known electron transfer, and mediator of apoptotic cell death may be involved in the oxidative stress-induced aggregation of alpha-synuclein in Parkinson's disease and related disorders.

关键词：乙酰半胱氨酸/药理学抗氧化剂/药理学脑/病理学脑/超微结构细胞色素c组/分析细胞色素c组/代谢去铁胺/药理学过氧化氢/药理学免疫组织化学 Lewy体病/代谢 Lewy体病/病理学神经组织蛋白质类/分析神经组织蛋白质类/代谢氧化性应激重组蛋白质类/代谢突触核蛋白类 α突触核蛋白人类

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Oxidative damage linked to neurodegeneration by selective α-synuclein nitration in synucleinopathy lesions

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SCIENCE 2000年第5493期290卷 985-989页

作者： Giasson, BI Duda, JE Murray, IVJ Chen, QP Souza, JM Hurtig, HI Ischiropoulos, H Trojanowski, JQ Lee, VMY Univ Penn Ctr Neurodegenerat Dis Res Philadelphia PA 19104 USA Univ Penn Dept Pathol & Lab Med Philadelphia PA 19104 USA Univ Penn Dept Neurol Philadelphia PA 19104 USA Childrens Hosp Philadelphia Dept Biochem & Biophys Philadelphia PA 19104 USA Stokes Res Inst Philadelphia PA 19104 USA

Aggregated alpha -synuclein proteins form brain Lesions that are hallmarks of neurodegenerative synucleinopathies, and oxidative stress has been implicated in the pathogenesis of some of these disorders. Using antibodies to specific nitrated tyrosine residues in alpha -synuclein, we demonstrate extensive and widespread accumulations of nitrated alpha -synuclein in the signature inclusions of Parkinson's disease, dementia with Lewy bodies, the Lewy body variant of Alzheimer's disease, and multiple system atrophy brains. We also show that nitrated alpha -synuclein is present in the major filamentous building blocks of these inclusions, as well as in the insoluble fractions of affected brain regions of synucleinopathies. The selective and specific nitration of alpha -synuclein in these disorders provides evidence to directly link oxidative and nitrative damage to the onset and progression of neurodegenerative synucleinopathies.

关键词：阿尔茨海默病/代谢阿尔茨海默病/病理学抗体单克隆印迹法蛋白质脑/代谢脑/病理学脑化学酶联免疫吸附测定荧光抗体技术免疫组织化学 Lewy体/化学 Lewy体病/代谢 Lewy体病/病理学显微镜检查免疫电子多系统萎缩/代谢多系统萎缩/病理学神经组织蛋白质类/分析神经组织蛋白质类/免疫学神经组织蛋白质类/代谢神经变性疾病/代谢神经变性疾病/病理学神经元/化学神经元/代谢神经元/超微结构氧化性应激帕金森病/代谢帕金森病/病理学突触核蛋白类酪氨酸/类似物和衍生物酪氨酸/分析酪氨酸/免疫学酪氨酸/代谢 α突触核蛋白人类

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Morphological changes in periodontal mechanoreceptors of mouse maxillary incisors after the experimental induction of anterior crossbite: a light and electron microscopic observation using immunohistochemistry for PGP 9.5

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EUROPEAN JOURNAL OF ORTHODONTICS 1999年第1期21卷 15-29页

作者： Piyapattamin, T Takano, Y Eto, K Soma, K Tokyo Med & Dent Univ Fac Dent Dept Orthodont 1 Bunkyo Ku Tokyo 1138549 Japan Tokyo Med & Dent Univ Dept Oral Anat 2 Tokyo 1138549 Japan Tokyo Med & Dent Univ Dept Dev Biol Tokyo 1138549 Japan

Ruffini nerve endings (mechanoreceptors) in the periodontal ligament (PDL) of mouse incisors were examined to elucidate whether experimentally-induced crossbites cause any changes or abnormalities in their morphology and distribution. Anterior guiding planes were attached to the mandibular incisors of 8-week-old C3H/HeSIc mice. At 3 days and 1, 2, 4, 6, and 8 weeks post-attachment of the appliance, the mice were sacrificed by perfusion fixation. Frozen sagittal cryostat sections of the decalcified maxillary incisors were processed for immunohistochemistry of protein gene product 9.5, followed by histochemical determination of tartrate-resistant acid phosphatase activity to reveal sites of alveolar bone resorption. Despite the absence of bone resorption within the lingual PDL of control mice, distinct resorption sites were seen in the respective regions of the experimental animals. Unlike the controls, many Ruffini endings showing vague and swollen contours, with unusually long and pedunculated micro-projections were observed in the affected lingual PDL of the incisors in the experimental animals with short-term anterior crossbite induction. Club-shaped nerve terminations with few, if any, micro-projections were observed in the lingual PDL of experimental animals with long-term induction, as well as in aged control mouse incisors. Differences in the distribution of Ruffini endings were also observed. These results indicate that changing the direction of the force applied to the PDL results in rapid and prolonged changes in the morphology of Ruffini-like mechanoreceptors.

关键词：酸性磷酸酶/分析牙槽骨质丢失/病理学方差分析生物学标记/分析咬合力免疫组织化学切牙/神经支配同工酶类/分析错|牙合|/病理学上颌骨/神经支配机械感受器/病理学机械感受器/超微结构小鼠近交C3H 小鼠近交系显微镜检查电子神经末梢/病理学神经末梢/超微结构神经组织蛋白质类/分析破骨细胞/病理学牙周膜/神经支配硫酯水解酶类/分析泛素硫酯酶动物小鼠

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Immunohistochemical analysis for neural markers of the lateral retinaculum in patients with isolated symptomatic patellofemoral malalignment -: A neuroanatomic basis for anterior knee pain in the active young patient

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AMERICAN JOURNAL OF SPORTS MEDICINE 2000年第5期28卷 725-731页

作者： Sanchis-Alfonso, V Roselló-Sastre, E Hosp Arnau Vilanova Dept Orthopaed Surg Valencia Spain Hosp Univ Dr Peset Dept Pathol Valencia Spain

We evaluated 13 lateral retinacula excised at the time of Insall proximal realignments or isolated lateral retinacular releases performed in patients with isolated symptomatic patellofemoral malalignment recalcitrant to nonoperative treatment. Evaluation was performed by means of conventional histologic and immunohistochemical analysis for neural markers (S-100 protein, neurofilament protein, substance P, and neural growth factor). The observations reported here provide a neuroanatomic basis for anterior knee pain syndrome in active young patients with isolated symptomatic patellofemoral malalignment and support the clinical observation that the lateral retinaculum may have a key role in the origin of this pain as a result of increased neural growth factor production, which induces proliferation of nociceptive axons, mainly in a perivascular location.

关键词：生物学标记/分析免疫组织化学膝关节/解剖学和组织学膝关节/神经支配膝关节/病理学神经组织蛋白质类/分析疼痛/病理生理学髌骨/畸形综合征青少年成年人女(雌)性人类男(雄)性

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Molecular dissection of regulated secretory pathways in human gastric enterochromaffin-like cells:: an immunohistochemical analysis

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HISTOCHEMISTRY AND CELL BIOLOGY 1999年第3期112卷 205-214页

作者： Höcker, M John, M Anagnostopoulos, J Buhr, HJ Solimena, M Gasnier, B Henry, JP Wang, TC Wiedenmann, B Humboldt Univ Klinikum Charite Med Klin Schwerpunkt Hepatol & Gastroenterol D-13353 Berlin Germany Free Univ Berlin Klinikum Benjamin Franklin Med Klin 1 D-12002 Berlin Germany Free Univ Berlin Klinikum Benjamin Franklin Abt Pathol D-12002 Berlin Germany Free Univ Berlin Klinikum Benjamin Franklin Allgemeinchirurg Abt D-12002 Berlin Germany Yale Univ Sch Med Div Med New Haven CT USA Yale Univ Sch Med Div Endocrinol New Haven CT USA Inst Biol Phys Chim F-75005 Paris France Harvard Univ Massachusetts Gen Hosp Sch Med Gastroenterol Unit Boston MA USA Harvard Univ Massachusetts Gen Hosp Sch Med Dept Internal Med Boston MA USA

Enterochromaffin-like (ECL) cells regulate gastric acid secretion through vesicular release of histamine. Until now, the molecular machinery of human ECL cells involved in the formation and release of vesicles is largely unknown. We analyzed tissue samples obtained from normal human gastric mucosa (n=4) and ECLomas (n=5) immunohistochemically using the APAAP method or double immunofluorescence confocal laser microscopy. Human pheochromocytomas (n=5) were investigated in parallel and compared to ECL cells. Secretory pathways were characterized using antibodies specific for marker proteins of large dense-core vesicles (LDCVs;islet cell antigen 512, chromogranin A, pancreastatin, and vesicular monoamine transporter 2) and small synaptic vesicle (SSV) analogues (synaptophysin). Tissues were also analyzed for expression of the peptide hormone processing enzymes, carboxypeptidase E and prohormone convertase 1, as well as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, 25-kDa synaptosome-associated protein (SNAP25), syntaxin, and syn aptobrevin. Immunoreactivity for markers of LDCVs and SSV analogues were detected in normal ECL cells and ECLomas. Both tissues also showed expression of carboxypeptidase E and prohormone convertase 1. Analysis of vesicular SNARE (v-SNARE) and target membrane SNARE (t-SNARE) proteins revealed the presence of SNAP25, syntaxin, and synaptobrevin in normal and neoplastic ECL cells. Our data suggest that ECL cells possess the two vesicle types of regulated neuroendocrine secretory pathways, LDCVs and SSV analogues. Since ECL cells also contain typical SNARE proteins, the molecular machinery underlying secretory processes in this cell type appears to be identical to the secretory apparatus of neuroendocrine cells and neurons. In addition, our findings suggest that the secretory apparatus of ECL cells is maintained during neoplastic transformation.

关键词：天冬氨酸内肽酶类/分析生物学标记/分析羧肽酶H 羧肽酶类/分析载体蛋白质类/分析细胞质颗粒/化学细胞质颗粒/酶学肠嗜铬样细胞/化学肠嗜铬样细胞/酶学肠嗜铬样细胞/病理学肠嗜铬样细胞/分泌荧光抗体技术间接胃黏膜/化学胃黏膜/酶学胃黏膜/病理学胃黏膜/分泌免疫酶技术膜蛋白质类/分析神经组织蛋白质类/分析嗜铬细胞瘤/化学嗜铬细胞瘤/酶学嗜铬细胞瘤/病理学嗜铬细胞瘤/分泌前蛋白转化酶类 Qa-SNARE蛋白质类 Qc-SNARE蛋白质类 R-SNARE蛋白质类胃肿瘤/化学胃肿瘤/酶学胃肿瘤/病理学胃肿瘤/分泌突触体相关蛋白质25 膜泡运输蛋白质类人类

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Radiation-induced apoptosis in the neonatal and adult rat spinal cord

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RADIATION RESEARCH 2000年第3期154卷 268-276页

作者： Li, YQ Wong, CS Univ Toronto Univ Hlth Network Princess Margaret Hosp Dept Radiat Oncol Toronto ON M5G 2M9 Canada Univ Toronto Div Expt Therapeut Toronto ON M5G 2M9 Canada

This study was designed to characterize radiation-induced apoptosis in the spinal cord of the neonatal and young adult rat. Spinal cords (C2-T2) of 1-, 2 and 10-week-old rats were irradiated with a single dose of 8, 18 or 22 Gy, Apoptosis was assessed histologically according to its specific morphological features or by using the TUNEL assay. Cell proliferation was assessed immunohistochemically using BrdU, Identities of cell types undergoing apoptosis were assessed using immunohistochemistry or in situ hybridization using markers for neurons, glial progenitor cells, microglia, oligodendrocytes and astrocytes, The time course of radiation-induced apoptosis in 1- or 2-week-old rat spinal cord was similar to that in the young adult rat spinal cord. A peak response was observed at about 8 h after irradiation, and the apoptosis index returned to the levels in nonirradiated spinal cords at 24 h, The neonatal rat spinal cord demonstrated increased apoptosis compared to the adult, Values for total yield of apoptosis over 24 h induced by 8 Gy in the neonatal rat spinal cord were significantly greater than that in the adult. Immunohistochemistry studies using Leu7, galactocerebroside, Rip and adenomatous polyposis coli tumor suppressor protein indicated that most apoptotic cells were cells of the oligodendroglial lineage regardless of the age of the animal. No evidence of Gfap or factor VIII-related antigen positive apoptotic cells was observed, and there was a small number of apoptotic microglial cells (lectin-Rca1 positive) in the neonatal and adult rat spinal cord. In the neonatal but not adult rat spinal cord, about 10% of the apoptotic cells appeared to be neurons and were immunoreactive for synaptophysin, Labeling indices (LI) for BrdU in nonirradiated 1- and 2-week-old rat spinal cord were 20.0 and 16.3%, respectively, significantly greater than the LI of 1.0% in the 10-week-old rat spinal cord. At 8 h after a single dose of 8 Gy, 13.4% of the apoptotic cells were Brd

关键词：年龄因素动物新生细胞凋亡/辐射效应细胞分裂/辐射效应因子Ⅷ/分析神经胶质原纤维酸性蛋白质/分析原位杂交原位缺口末端标记神经组织蛋白质类/分析神经胶质/病理学神经胶质/辐射效应神经元/病理学神经元/辐射效应辐射损伤实验性/病理学辐射耐受性大鼠近交F344 脊髓/生长和发育脊髓/病理学动物女(雌)性大鼠

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POPULATION PARAMETERS - ACQUISITION AND ANALYSIS OF ELECTROPHORETIC BRAIN PROTEIN PATTERNS BY MINICOMPUTER-COUPLED DENSITOMETRY

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ANALYTICAL BIOCHEMISTRY 1975年第2期69卷 350-360页

作者： MAYOR, SJ MED COLL OHIO DEPT PHYSIOLTOLEDOOH 43614

Interfacing a minicomputer with a high resolution densitometer permits the online acquisition and statistical analysis of population parameters for experimental and control sample patterns of various macromolecules obtained by polyacrylamide gel electrophoresis. This technique, illustrated in this paper using protein from the brains of Japanese quail, enables the experimenter to make comparisons which were previously not readily available for electrophoresis.

关键词：脑化学计算机光密度测定法/方法电泳聚丙烯酰氨凝胶/方法神经组织蛋白质类/分析鹌鹑动物男(雄)性

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Evidence that endogenous thymosin alpha-1 is present in the rat central nervous system

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NEUROCHEMISTRY INTERNATIONAL 1999年第6期35卷 463-470页

作者： Turrini, P Aloe, L CNR Inst Neurobiol I-00137 Rome Italy

We have previously reported that administration of Thymosin alpha 1 (T-alpha 1) can enhance the level of the Nerve Growth Factor and the distribution of its receptor in the developing Central Nervous System (CNS) of rat. To further explore the role of T-alpha 1 and verify its presence in cells of rat CNS, we carried out an immunohistochemical study using a polyclonal antibody against T-alpha 1. T-alpha 1 immunoreactivity was found mainly in neurons of the hippocampus and spinal cord and in several small cells, resembling glial cells, of specific regions of the brain. Moreover, to study whether cerebral cells were receptive to T-alpha 1, we injected iodinated T-alpha 1 (I-125-T-alpha 1) icv. I-125-T-alpha 1 labelled neurons were observed in the hypothalamus and septal nuclei. Our results indicate that specific neuronal populations in the rat CNS are able to express and respond to T-alpha 1. (C) 1999 Elsevier Science Ltd. All rights reserved.

关键词：脑化学齿状回/化学荧光抗体技术间接海马/化学免疫血清免疫酶技术运动神经元/化学神经组织蛋白质类/分析神经胶质/化学神经免疫调节神经元/化学锥体细胞/化学大鼠 Sprague-Dawley 隔核/化学脊髓/化学胸腺素/类似物和衍生物胸腺素/分析胸腺素/免疫学胸腺素/药代动力学动物女(雌)性男(雄)性兔大鼠

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