胚鼠室管膜及成鼠骨髓神经干细胞分离培养及分化鉴定实验研究

目的 :探寻胚鼠室管膜和成鼠骨髓分离培养神经干细胞 (NSC)的可行性。方法 :将分离的胚脑室管膜组织或成年骨髓细胞分别特殊培养。以细胞克隆及免疫细胞化学方法判断NSC增殖并鉴定NSC、神经元和神经胶质细胞。结果 :两种组织来源细胞在相应培养条件下NSC均有快速增殖。可得到具有长突起并建立有网状联系的神经元及胶质细胞。结论 :由胚鼠室管膜和成鼠骨髓诱导分化NSC是可行的     

The absence of Complexin 3 and Complexin 4 differentially impacts the ON and OFF pathways in mouse retina

Complexins (Cplxs) regulate the speed and Ca₂₊‐sensitivity of synaptic vesicle fusion. It has been shown that all four known Cplxs are present at mouse retinal synapses – at conventional amacrine cell synapses (Cplx 1 to Cplx 3) and at photoreceptor and bipolar cell ribbon synapses (Cplx 3 and Cplx 4) [ K. Reim et al. (2005) J. Cell Biol., 169 , 669‐680]. Electroretinographic recordings in Cplx 3/Cplx 4 double‐knockout (DKO) mice showed perturbed transmission in the outer plexiform layer, and possible changes in the inner plexiform layer [ K. Reim et al. (2009) J. Cell Sci., 122 , 1352–1361]. In the present study, we examined the effects of the absence of Cplx 3 and Cplx 4 on ganglion cell responses. We report that the lack of Cplx 3 and Cplx 4 differentially impacts the ON and OFF pathways. Under photopic conditions, the responses in the cone OFF pathway are largely unaffected, whereas the responses in the cone ON pathway are diminished in Cplx 3/Cplx 4 DKO mice. Under scotopic conditions, both ON and OFF response rates are reduced and high‐sensitivity OFF responses are missing in Cplx 3/Cplx 4 DKO mice. The electrophysiological findings are corroborated by new immunocytochemical findings. We now show that rod spherules contain only Cplx 4. However, both Cplx 3 and Cplx 4 co‐localize in cone pedicles. In the inner plexiform layer, Cplx 3 is present in rod bipolar cell terminals and in amacrine cell processes. Most importantly, Cplx 3 is localized in the lobular appendages of AII amacrine cells, the sites of signal transmission from the primary rod pathway into the OFF pathway in the inner plexiform layer.     

Ultrastructural study of the granule cell domain of the cochlear nucleus in rats: Mossy fiber endings and their targets

The principal projection neurons of the cochlear nucleus receive the bulk of their input from the auditory nerve. These projection neurons reside in the core of the nucleus and are surrounded by an external shell, which is called the granule cell domain. Interneurons of the cochlear granule cell domain are the target for nonprimary auditory inputs, including projections from the superior olivary complex, inferior colliculus, and auditory cortex. The granule cell domain also receives projections from the cuneate and trigeminal nuclei, which are first-order nuclei of the somatosensory system. The cellular targets of the nonprimary projections are mostly unknown due to a lack of information regarding postsynaptic profiles in the granule cell areas. In the present paper, we examined the synaptic relationships between a heterogeneous class of large synaptic terminals called mossy fibers and their targets within subdivisions of the granule cell domain known as the lamina and superficial layer. By using light and electron microscopic methods in these subdivisions, we provide evidence for three different neuron classes that receive input from the mossy fibers: granule cells, unipolar brush cells, and a previously undescribed class called chestnut cells. The distinct synaptic relations between mossy fibers and members of each neuron class further imply fundamentally separate roles for processing acoustic signals. © 1996 Wiley-Liss, Inc.     

SD大鼠胚胎神经干细胞分离、培养及鉴定

目的 体外培养并鉴定神经干细胞，为相关实验研究奠定基础。方法 分离SD大鼠胎鼠的间脑，加入神经生长因子EGF和bFGF在神经干细胞条件培养基中克隆培养。用免疫细胞化学方法鉴定分离的神经干细胞。结果 分离培养的细胞具有不断增殖的能力，表达神经巢蛋白（nestin），并能经过诱导分化为神经元和神经胶质细胞。结论 成功建立了神经干细胞的分离培养方法，可用于进一步的实验研究。     

Thymic innervation in the rat: A light and electron microscopical study

The innervation of the rat thymus was studied by light and electron microscopy in juvenile and aged rats. By light microscopy numerous fine nerves were found in the connective tissue septa penetrating between the thymic lobules. These septa were clearly delineated in the juvenile animals, but indistinct in the aged rats, thus creating the spurious impression that thymic parenchyma contains nerves. In the aged animals the nerves are thicker, tortuous, and more branched than in juvenile animals. Electron microscopy confirms the light microscopic observations: no nerves were found within the thymic parenchyma. The thymic capsule and larger connective tissue septa contain bundles of myelinated and unmyelinated axons, surrounded by a perineural sheath. Within the extraparenchymal compartment, which is greatly enlarged in aged animals, efferent and sensory nerves, devoid of perineurium, were found to contact mainly reticular cells, and in rare instances plasma cells and lymphocytes. The majority of axonal varicosities are not closely related to cellular elements, and, in general, vesicles are relatively infrequent. The possible functional significance of these observations is discussed.     

诱导多能干细胞与神经退行性疾病(英文)

神经退行性疾病,包括帕金森病、阿尔茨海默病和肌萎缩侧索硬化症等的共同特征是在中枢神经系统的不同部位发生特发性神经元丢失。这些神经元的丢失给病人造成了一系列相应的功能障碍。应用人类胚胎干细胞进行细胞替代治疗曾引起人们很大的兴趣,但是一些伦理学问题阻碍了该研究的发展。通过导入特定的转录因子,体细胞能够被诱导为具有胚胎干细胞特性的细胞,即诱导多能干细胞(induced pluripotent stem cells, iPS cells)。获取人类iPS细胞并不涉及明显的伦理问题,并且运用病人特异性的iPS细胞能使自体移植成为可能。因此,iPS细胞有可能成为细胞替代治疗中可靠的细胞来源。此外,利用iPS细胞,人们还能在体外直接研究病变神经细胞的表型以及神经细胞在特定致病因子作用下的疾病易感性,有助于揭示神经退行性疾病的内在机制。本文综述了iPS细胞用于神经退行性疾病细胞治疗的最新进展,并探讨了其在建立疾病的细胞模型中的潜在价值。     

肿瘤干细胞的分离与纯化研究进展

随着肿瘤干细胞学说的日趋成熟,针对干细胞的靶向治疗已成为新的理论,分离与纯化肿瘤干细胞是研究的重要组成部分。目前国内外主要根据肿瘤干细胞已知的细胞表型和生物学特性,用流式细胞仪分选法、磁性激活细胞分选法和旁群细胞分选法来分离与纯化肿瘤干细胞,为肿瘤的临床诊断与治疗提供了依据。     

Isolation, culture, and characterization of adult rat oligodendrocytes.

Investigation into CNS demyelinating diseases, which usually occur in adults, can be facilitated by the use of a good in vitro model. We have established a methodology whereby oligodendrocytes from adult rat CNS can be cultured in vitro, and we have characterized these cultures morphologically and immunologically. Approximately 1 g of spinal cord and brainstem per adult rat was removed and dissociated mechanically and enzymatically. After filtration of the white matter homogenate, myelin was removed by 0.9 M sucrose density centrifugation. The cells were further purified by centrifugation through a 0.3%/4% discontinuous gradient of bovine serum albumin (BSA). The pellet was resuspended and placed in an untreated 6-well culture dish overnight to allow the astrocytes to attach. The non-adherent cells were replated on poly-l-lysine-treated coverslips. Approximately 8.25 x 10(5) cells were recovered per animal. The adult oligodendrocytes initially appeared as rounded cell bodies, but after 2-5 days in vitro (DIV), the oligodendrocytes extended 6-10 thick processes. A membrane sheath between these processes was immunostainable with either anti-galactocerebroside (GC), anti-O4, anti-myelin basic protein (MBP), or anti-2'3' cyclic nucleotide 3' phosphohydrolase (CNPase) and was also evident in scanning EM. Older cultures (up to 60 DIV) maintained whorls of myelin and transmission EM revealed a major dense line distance of approximately 103 A with up to 11 concentric layers of membrane. Immunologically, the adult oligodendrocytes are GC+, O4+, MBP+, CNPase+, and GFAP-. The method described will allow adult rat oligodendrocytes to be isolated and maintained in culture; these cultures retain the characteristics of differentiated adult oligodendrocytes.     

神经干细胞诱导分化神经元培养方法的建立

目的:诱导大鼠神经干细胞(NSCs)向神经元细胞系分化,建立体外培养神经元的模型。方法:从Wistar大鼠胚胎大脑分离、培养NSCs,小剂量碱性成纤维生长因子(bFGF)无血清条件培养基诱导NSCs定向分化,以免疫组化技术检测,与直接培养的海马细胞中NF200阳性细胞数以及神经元生长情况进行比较。结果:应用小剂量bFGF无血清培养基诱导NSCs定向分化7、10d,神经元生长良好,通过免疫组化检测到NF200阳性神经元数量多于直接培养的海马神经元(P<0.01)。结论:应用小剂量bFGF无血清条件培养基对NSCs进行定向诱导分化,培养的神经元生长良好,可作为体外神经元培养模型之一。     

心肌条件培养液与5-氮胞苷诱导骨髓基质干细胞向心肌样细胞的分化

背景：骨髓基质干细胞可以明显改善心肌缺血时的心脏功能，但其直接分化为心肌细胞并参与心功能恢复的机制尚不明确。 目的：探讨心肌条件培养液与5-氮胞苷诱导骨髓基质干细胞分化为心肌样细胞的作用。 方法：全骨髓贴壁法分离培养骨髓基质干细胞，将第6代骨髓基质干细胞随机分为4组：5-氮胞苷+心肌条件培养液联合诱导组；5-氮胞苷组；心肌条件培养液组；基础培养基组(空白组)。用心肌条件培养液和5-氮胞苷诱导3周后用免疫组化法测定各组心肌肌钙蛋白表达，并采用单因素方差分析检验进行统计学分析。 结果与结论：在体外心肌条件培养液可以诱导骨髓基质干细胞向心肌细胞分化，提示其中细胞因子可能起关键作用，但心肌条件培养液的这种作用要弱于化学诱导剂5-氮胞苷的诱导作用，且联合诱导作用更强。     

脑动静脉畸形血管内皮细胞和平滑肌细胞的体外培养

目的：探讨脑动静脉畸形（AVM）内皮细胞和平滑肌细胞的分离、培养及鉴定方法。方法：手术获得脑AVM标本后,分别采用组织块贴壁法和酶消化法对脑AVM血管内皮细胞、平滑肌细胞进行培养和形态学观察,采用免疫组化分别检测培养的内皮细胞CD31抗原和平滑肌细胞SMA抗原阳性表达。结果：相差显微镜下,培养的内皮细胞呈扁平梭形,胞核椭圆居中;平滑肌细胞呈长梭形。CD31和SMA分别在两种细胞中免疫阳性表达率超过90％。结论：AVM内皮细胞、平滑肌细胞可以获取和培养增殖,为可供研究脑血管畸形血管生物学的体外模型。     

Overlap in the distribution of cholinergic and catecholaminergic neurons in the upper brainstem of the ferret

The distribution of catecholaminergic and cholinergic neurons in the upper brainstem of the ferret were mapped by staining immunohistochemically two adjacent series of sections of brainstem for tyrosine hydroxylase and choline acetyltransferase, respectively. As in other species, large numbers of tyrosine-hydroxylase-positive neurons are localized in the ventral tegmental area (A10), the substantia nigra (A9), and in A8. Tyrosine-hydroxylase-positive neurons in the dorsolateral pontine tegmentum (A4, A6, and A7--the locus coeruleus complex) of the ferret are rather diffusely distributed, as has been observed in other carnivore species such as the cat and the dog, but unlike the cat, these cells in the ferret display a relative uniformity in size and morphology. Choline-acetyltransferase-positive neurons which extend in the ferret's pedunculopontine tegmental nucleus and ventral parabrachial area (Ch5) are relatively large cells that stain intensely for choline acetyltransferase, and their dendrites form prominent bundles in regions where unstained fibre tracts are prevalent. Choline-acetyltransferase-positive neurons distributed in the laterodorsal tegmental nucleus (Ch6) are smaller than the cholinergic cells of Ch5, and they stain less intensely for choline acetyltransferase. Rostrally, there is little overlap between the catecholaminergic cell groups A8, A9, and A10 and the cholinergic cell groups of Ch5 and Ch6. Caudally, the Ch5 neurons extend some considerable extent into the locus coeruleus complex. In the region of overlap, no cells with staining for both tyrosine hydroxylase and choline acetyltransferase were observed, as was ascertained with a double staining method employing a combination of tyrosine hydroxylase immunofluorescence and choline acetyltransferase peroxidase-antiperoxidase immunohistochemistry. In conclusion, the ferret has a typically carnivore pattern for the distribution of catecholaminergic cells in the upper brainstem, and there is a significant overlap between the catecholaminergic and cholinergic cell groups in the dorsolateral pontine tegmentum.     

Growth and radiosensitivity of irradiated human glioma cell progeny

BACKGROUND： Progenitors of the immortalized human glioma cell line, SHG-44, are significantly less sensitive to irradiation. Two hypotheses regarding the mechanism of this effect exist： several studies have suggested that there is a subgroup with different radiosensitivities in identical cell group, and the progenitors of irradiate is a adaptive response subgroup, so its radiosensitivity is descend. A second hypothesis suggests that irradiated glioma progeny have a stronger ability to repair DNA damage. This would suggest that when progeny are continuously irradiated, resistance to irradiation-induced DNA increases, and radiosensitivity decreases. OBJECTIVE： To investigate radiosensitivity and growth features after irradiation to progeny of the human glioma cell line SHG-44. DESIGN, TIME AND SETTING： A randomized, controlled experiment, which was performed at the Department of Radiology Laboratory, the First Hospital Affiliated to Soochow University, between September 2004 and January 2006. MATERIALS： The glioma cell line SHG-44 was provided by the Institute of Neuroscience, First Affiliated Hospital of Suzhou University. Propidium iodide reagent was provided by Coulter Corporation. A linear accelerator, KD-2 type, was provided by Siemens, Germany. The flow cytometer EPICS-XL was provided by Coulter Corporation. METHODS： Brain glioma SHG-44 cells were divided into four groups： SHG-44, SHG-44-2, SHG-44-6, and SHG-44-10 . The SHG-44-2, SHG-44-6, and SHG-44-10 cells were vertically irradiated with varying doses of 2, 6 and 10 Gy by a linear accelerator （6 MVX）. The cells were passaged for 15 generations and cultured in RPMI-1640 culture media. MAIN OUTCOME MEASURES： Community re-double time, mean lethal dose （D0）, extrapolation number （N）, fraction surviving fraction irradiated by 2 Gy dose （SF2）, quasi-threshold dose （Dq）, and cell cycle. RESULTS： The Population doubling time （PDT） of SHG-44-2, SHG-44-6, and SHG-44-10 cell groups was not significant （P = 0.052）. Compare     

bFGF、EGF诱导成人骨髓基质细胞向血管内皮细胞分化的实验研究

目的探讨骨髓基质细胞向内皮细胞分化的可能性。方法从成人肋骨中分离培养骨髓基质细胞，用bFGF和EGF作为诱导剂，观察细胞形态的变化，采用免疫组织化学方法检测诱导后的细胞表达血管相关性Ⅷ情况，并观察体外微血管网状结构的形成情况。结果经bFGF和EGF诱导后的细胞在形态上表现为内皮细胞特征，血管相关性Ⅷ呈阳性表达，并且在体外可形成微血管样结构。结论骨髓基质细胞在bFGF和EGF诱导下，可以分化为内皮细胞。     

Follicle and melanocyte stem cells, and their application in neuroscience A Web of Science-based literature analysis

OBJECTIVE: To identify global research trends of follicle and melanocyte stem cells, and their application in neuroscience. DATA RETRIEVAL: We performed a bibliometric analysis of studies from 2002 to 2011 on follicle and melanocyte stem cells, and their application in neuroscience, which were retrieved from the Web of Science, using the follicle stem cell or melanocyte stem cell, and neural, neuro or nerve. SELECTION CRITERIA: Inclusion criteria: (a) peer-reviewed published articles on follicle and melanocyte stem cells, and their application in neuroscience, which were indexed in the Web of Science; (b) original research articles, reviews, meeting abstracts, proceedings papers, book chapters, editorial material, and news items. Exclusion criteria: (a) articles that required manual searching or telephone access; (b) documents that were not published in the public domain; and (c) a number of corrected papers from the total number of articles. MAIN OUTCOME MEASURES: (1) Distribution of publications on follicle and melanocyte stem cells by years, journals, countries, institutions, institutions in China, and most cited papers. (2) Distribution of publications on the application of follicle and melanocyte stem cells in neuroscience by years, journals, countries, institutions, and most cited papers. RESULTS: Of the 348 publications from 2002 to 2011 on follicle and melanocyte stem cells, which were retrieved from the Web of Science, more than half were from American authors and institutes. The most prolific institutions in China for publication of papers on follicle and melanocyte stem cells were the Fourth Military Medical University and Third Military Medical University. The most prolific journals for publication of papers on follicle and melanocyte stem cells were the Journal of Investigative Dermatology, Pigment Cell & Melanoma Research. Of the 63 publications from 2002 to 2011 on the application of follicle and melanocyte stem cells in neuroscience, which were retrieved from the Web of Science, more than half were from American authors and institutes, and no papers were from Chinese authors and institutes. The most prolific journals for publication of papers on the application of follicle and melanocyte stem cells in neuroscience were the Journal of Investigative Dermatology, Pigment Cell & Melanoma Research. CONCLUSION: Based on our analysis of the literature and research trends, we found that follicle stem cells might offer further benefits in neural regenerative medicine.     

Growth and death of cells of the mesencephalic fifth nucleus in Rana pipiens larvae

Positions, numbers, and nuclear sizes of mesencephalic fifth nucleus M-V) cells were determined in Rana pipiens larvae in stages XIII through the end of metamorphosis at stage XXV, in a few just postmetamorphic juveniles, and in adults. M-V cells are found throughout the tectum, with the greatest concentrations just anterior to each optic ventricle, and along the medioanterior aspect of each ventricle. Some cells lie below these levels, adjacent to the ependyma of the expanded aqueduct, the mesencephalic ventricle; a very few cells occur in the anterior medullary velum and in the adjacent anterior cerebellum. Up to 879 M-V cells were seen in single animals in stages XIV-XVIII, with mean values near 650 cells. Pyknotic M-V cells appear in all stages from XVII through XXV, and possibly in the youngest juveniles. Peak rates of cell loss occur at stage XXI, some 4-5 days after the forelimbs have emerged. Mean postmetamorphic cell numbers are about 350, suggesting an average M-V cell loss of 300 per animal. Nuclear cross-sectional areas are 72 microns2 at stage XIII, slightly above the size of such cells in hypophysectomized tadpoles. Nuclear growth is progressive to 102-111 microns2 at stages XXI-XXV. Adult sizes increase to a mean of 121 microns2. Hypophysectomized animals display pyknotic cells only after the tadpoles have been strongly stimulated by thyroid hormone, as demonstrated by significant growth of the legs and of the M-V cells themselves.     

精神分裂症患者脑脊液T,B细胞标记及IgG合成率研究

应用ＡＮＡ冰霜示记Ｔ细胞，ＤＡＫＯ－ＣＤ２０单克隆抗体ＡＢＣ法标记Ｂ细胞，观察了３２例精神分裂症患者脑脊液中Ｔ、Ｂ的异常变化，并应用酶联免疫荧光法测定了其中１３例患者中枢神经系统的ＩｇＧ合成率。实验结果提示，精神分裂症患者Ｔ细胞百分率偏低，而Ｂ细胞百分率明显偏高，有５３．８４％的患者中枢神经系统有内源性ＩｇＧ的异常合成。     

Association of the AMPA receptor-related postsynaptic density proteins GRIP and ABP with subsets of glutamate-sensitive neurons in the rat retina

We used specific antibodies against two postsynaptic density proteins, GRIP (glutamate receptor interacting protein) and ABP (AMPA receptor-binding protein), to study their distribution in the rat retina. In the central nervous system, it has been shown that both proteins bind strongly to the AMPA glutamate receptor (GluR) 2/3 subunits, but not other GluRs, through a set of three PDZ domains. Western blots detected a single GRIP protein that was virtually identical in retina and brain, whereas retinal ABP corresponded to only one of three ABP peptides found in brain. The retinal distributions of GluR2/3, GRIP, and ABP immunoreactivity (IR) were similar but not identical. GluR2/3 immunoreactivity (IR) was abundant in both plexiform layers and in large perikarya. ABP IR was concentrated in large perikarya but was sparse in the plexiform layers, whereas GRIP IR was relatively more abundant in the plexiform layers than in perikarya. Immunolabel for these three antibodies consisted of puncta < or = 0.2 microm in diameter. The cellular localization of GRIP and ABP IR was examined by double labeling subclasses of retinal neuron with characteristic marker proteins, e.g., calbindin. GRIP, ABP, and GluR2/3 IR were detected in horizontal cells, dopaminergic and glycinergic AII amacrine cells and large ganglion cells. Immunolabel was absent in rod bipolar and weak or absent in cholinergic amacrine cells. By using the tyramide method of signal amplification, a colocalization of GluR2/3 was found with either GRIP or ABP in horizontal cell terminals, and perikarya of amacrine and ganglion cells. Our results show that ABP and GRIP colocalize with GluR2/3 in particular subsets of retinal neuron, as was previously established for certain neurons in the brain.