低氧联合GDNF诱导大鼠神经干细胞分化为TH阳性神经元的实验研究

目的本试验在低氧环境下利用胶质源性神经营养因子(GDNF)对大鼠胚胎神经干细胞(NSCs)进行定向诱导分化,从而研究低氧对NSCs向多巴胺能神经元分化的影响,并获得相当数量的多巴胺能细胞,为下一步的细胞移植奠定基础。方法分离培养孕14 d～16 d Wistar大鼠胚胎皮质NSCs,通过传代培养获得单克隆生长的NSCs,在不同氧浓度下利用GDNF对大鼠NSCs进行定向诱导分化,通过免疫细胞化学方法检测TH阳性细胞表达。结果低氧可明显促进NSCs向TH阳性神经元分化,与常氧对照组比较有显著差异(P 0.01),且在3%低氧浓度时,NSCs向TH阳性神经元分化率最高。结论低氧可促进NSCs向TH阳性神经元分化,3%低氧浓度是促进NSCs向TH阳性神经元分化的最适氧浓度,低氧促进NSCs向TH阳性神经元分化的分化率与氧浓度梯度无依赖关系,低氧联合GDNF诱导NSCs分化为TH阳性神经元,诱导效能更为显著。     

外源性Ang Ⅱ诱导神经干细胞向多巴胺能神经元分化的研究

目的在体外成功分离培养并扩增神经干细胞（NSCs）的基础上,研究外源性血管紧张素Ⅱ（Ang Ⅱ）对NSCs向多巴胺（DA）能神经元分化的影响。方法（1）分离培养新生1d SD大鼠脑组织NSCs,免疫细胞学方法测定NSCs特异性标志物神经上皮干细胞蛋白（Nestin）表达及其分化为神经元、神经胶质细胞的能力;（2）按培养液中Ang Ⅱ的浓度不同,分5个浓度（100、200、400、600、800nmol/L）对第二代NSCs进行诱导分化。10d后采用免疫细胞学方法检测DA能神经元标志物酪氨酸羟化酶（TH）和神经胶质细胞标志物胶质纤维酸性蛋白（GFAP）的表达,半定量RT-PCR（SQ-PCR）测定分化细胞中TH mRNA的相对表达量。结果外源性Ang Ⅱ诱导提高NSCs向TH阳性细胞分化的比率,TH mRNA相对表达量亦增加,以Ang Ⅱ浓度为400nmol/L及600nmol/L的诱导效果最明显,TH阳性细胞率分别为10.77%和11.34%,TH mRNA相对表达量分别为（0.4023±0.0515）和（0.3971±0.0319）,两组间比较无统计学意义（P〉0.05）;其中400nmol/L Ang Ⅱ组分化细胞中GFAP阳性细胞率高于对照组,有统计学意义（P〈0.05）。结论外源性AngⅡ促进NSCs向DA能神经元分化,在400～600nmol/L浓度范围内AngⅡ的诱导效能更显著;AngⅡ促进NSCs向DA能神经元分化的机制可能与同时促进星型胶质细胞（AS）分化有关。     

GDNF和GM1体外联合诱导骨髓基质干细胞向多巴胺能神经元分化的实验研究

目的应用GDNF和GM1体外联合诱导MSCs转化为多巴胺能(DA)神经元。探索体外诱导MSCs定向分化为DA能神经元的最佳条件。方法取雄性Wistar大鼠股骨和胫骨骨髓,密度梯度离心法分离获取单个核细胞。进行MSCs的体外培养和传代扩增。采用贴壁培养法使MSCs得到纯化。依据加入的神经营养因子不同分为对照组及实验组(GM1组、GDNF组、GDNF GM1组)。诱导过程中在倒置显微镜下观察细胞形态变化,分别在诱导第3天、第7天进行NSE、GFAP、TH免疫细胞化学检测。计数NSE和TH阳性细胞数,并计算阳性细胞百分比。采用SPSS10.0软件进行统计学处理。结果对照组可见少量NSE阳性细胞。各实验组比较发现,GDNF GM1组NSE阳性细胞率最高,GDNF组次之,GM1组最低。单独应用GDNF和GM1不能诱导MSCs表达TH,联合应用GDNF和GM1可诱导MSCs表达TH。随着诱导时间的延长,TH阳性表达增加。结论GDNF能够单独诱导MSCs向神经元样细胞分化,GM1不能单独诱导MSCs分化为神经元样细胞,但与GDNF联合,不仅可明显促进MSCs向神经元样细胞分化,而且部分细胞能够表达TH。     

胶质细胞源性神经营养因子诱导骨髓基质细胞向多巴胺能神经元分化的观察

目的 探讨胶质细胞源性神经营养因子（GDNF）在体外能否诱导骨髓基质细胞（BMSCs）向多巴胺（DA）能神经元分化及可能机制。方法无菌条件下，抽取成年SD大鼠胫骨内骨髓组织，分离制备成单细胞悬液进行培养。将增殖传代至第5代的BMSCs随机分为GDNF诱导组和对照组。继续培养7d后，应用BrdU／GFAP、BrdU/NeuN和TH免疫荧光单标和双标技术检测BMSCs增殖和分化情况。结果两组BMSCs继续培养7d后，增殖仍然活跃，有部分细胞向神经元和胶质样细胞分化，呈Brdu，GFAP、BrdU/NeuN和TH阳性表达，但GDNF组的增殖力更强，向神经元和TH神经元分化的数量明显多于对照组（P〈0．05）。结论GDNF能促进BMSCs的增殖和诱导BMSCs分化成神经元和胶质样细胞，其中少部分可分化为TH神经元（即DA能神经元）。     

骨髓源性神经干细胞诱导分化为多巴胺能神经元的实验研究

目的探索骨髓基质细胞(BMSCs)诱导分化为神经干细胞及多巴胺能神经元的分化条件和发生机制,比较不同血清浓度、不同白介素-1(IL-1α)及不同胶质细胞源神经营养因子(GDNF)浓度及不同组合浓度等诱导条件下BMSCs分化情况;为BMSCs在神经科学领域内的应用奠定基础。方法以成年SD大鼠BMSCs为实验对象,利用IL-1α、胶质细胞系来源GDNF等作为增殖及分化诱导因子,进行增殖培养、分化诱导;免疫细胞法进行细胞性质鉴定。结果加入GDNF、IL-1 α增殖培养诱导6 d,部分细胞有神经巢蛋白成分表达;三周测出 DA受体D2检测阳性,GDNF IL-1α组与 GDNF组及IL—1α组比较分化率更加显著(P<0．05)。结论 BMSCs在体外培养条件下,联合GDNF 和IL-1α诱导分化并配合使用高浓度血清(10％)可获得神经巢蛋白阳性的神经前体细胞及表达D2受体阳性的多巴胺能神经元;骨髓源性神经干细胞可以诱导分化为多巴胺能神经元。     

GFP-Nurr1基因修饰神经干细胞的建立及过表达Nurr1对神经干细胞向多巴胺神经元分化的影响

目的利用慢病毒载体建立GFP-Nurr1基因修饰的原代神经干细胞(NSCs)模型并观察Nurr1过表达后NSCs向多巴胺神经元的分化影响。方法利用基因重组构建pLenO-DCE-Nurr1慢病毒载体,用慢病毒转染第三代NSCs,转染72 h后荧光检测转染效果;设置空白对照组、空载体组及DCE-Nurr1组,分别用Western blot及PCR检测Nurr1的表达差异;并将转染后的NSCs分化培养7 d后分别用免疫细胞化学检测、Western blot及PCR检测酪氨酸羟化酶(TH)的表达。结果慢病毒转染NSCs 72 h后,转染率可达90%,与对照组相比DCE-Nurr1组高表达Nurr1。经慢病毒载体感染后的NSCs仍具备分化潜能,分化培养后发现DCE-Nurr1组分化的神经细胞中TH阳性细胞分化率90.60%,对照组为21.2%。结论慢病毒载体可高效转染NSCs过表达Nurr1;Nurr1基因过表达可以促进中脑腹侧来源NSCs向TH阳性多巴胺能神经元方向分化。     

血管紧张素Ⅱ体外诱导神经干细胞分化为多巴胺能神经元的机制研究

目的探讨血管紧张素II(AngiotensinII,AII)诱导神经干细胞(Neural stem cells,NSCs)定向分化为多巴胺(Dopamine,DA)能神经元的过程中血管紧张素1型(AT1)受体和血管紧张素2型(AT2)受体所起的作用。方法在无血清培养基中分离培养NSCs,通过巢蛋白(nestin)免疫细胞化学对神经前体细胞进行鉴定;在此基础上,将第二代的NSCs在含10%胎牛血清的培养基中按照实验设计分为6组,分别是,A:对照组,B:AII组,C:AT1受体拮抗剂ZD7155组,D:AT1受体拮抗剂ZD7155+AII组,E:AT2受体拮抗剂PD123319组,F:AT2受体拮抗剂PD123319+AII组,观察NSCs向DA能神经元定向诱导分化情况。通过酪氨酸羟化酶(TH)免疫细胞化学进行DA能神经元鉴定,并观察各组DA能神经元突触的长度变化,通过实时荧光定量RT-PCR检测各实验组中TH基因表达水平。结果细胞团中可以看到nestin免疫阳性着色,实时荧光定量RT-PCR法检测B组、D组的TH基因表达水平高于对照组,差异有统计学意义(P<0.05),C、E、F组的TH基因表达水平与对照组比较...     

GDNF及dbcAMP对神经干细胞诱导分化为多巴胺能神经元作用的实验研究

目的利用GDNF及dbcAMP对大鼠胚胎神经干细胞进行定向分化,从而获得相当数量的多巴胺能细胞,为下一步的细胞移植打下基础。方法分离培养孕14~16 d Wistar大鼠大脑神经干细胞,通过传代培养来检测其自我更新能力,并对其进行Nestin及Brdu蛋白鉴定,利用GDNF及dbcAMP对大鼠神经干细胞进行定向分化,通过免疫细胞化学及免疫荧光等方法检测TH阳性细胞表达。结果培养的神经干细胞均表达Nestin及Brdu,体外培养1 w后神经球的数量显著增加,体积增大;GDNF及dbcAMP均能提高TH表达,与血清对照组比较有显著差异(P0.05)。结论在神经营养因子的作用下,分离的神经干细胞能够快速增殖并形成神经球,GDNF及dbcAMP均有诱导神经干细胞向多巴胺能神经元分化的作用,其中GDNF在20μg/L时诱导效能最高,dbcAMP在200μmol/L时诱导效能最高,二者均存在剂量依赖性,二者联合诱导可明显提高各自单独诱导TH细胞的阳性率。     

体外中脑微环境下骨髓基质干细胞向多巴胺能神经元分化的实验研究

目的 应用中脑条件培养基与细胞因子联合诱导骨髓间质干细胞(MSCs)转化为多巴胺能(DA)神经元,探索体外诱导MSCs定向分化为DA能神经元的最佳条件.方法 取雄性Wistar大鼠股骨和胫骨骨髓,进行MSCs的体外培养和传代扩增.采用贴壁培养法使MSCs得到纯化.碱性成纤维生长因子(bFGF)预诱导后,依据加入的神经营养因子不同分为对照组及实验组[单唾液酸四己糖神经节苷脂(GM1)组、胶质源性神经营养因子(GDNF)组、GDNF+GM1组、GDNF+GM1+中脑条件培养基组].诱导过程中在倒置显微镜下观察细胞形态变化,分别在诱导第3、7天进行神经元特异烯醇化酶(NSE)、胶质纤维酸生蛋白(GFAP)、酪氨酸羟化酶(TH)免疫细胞化学检测.计数NSE和TH阳性细胞数,并计算阳性细胞百分比.结果 各实验组于诱导第3、7天见不同数量的NSE、TH阳性神经元样细胞,GFAP阴性.对照组及实验各组7 d时NSE阳性细胞数(个/视野,n=10)分别为:对照组2.214±0.779,GM1组22.014±3.624,GDNF组31.345±2.850,GDNF+GM1组40.314±4.203,GDNF+GM1+中脑条件培养基组45.257±5.999.实验各组以GDNF+GM1+中脑条件培养基组NSE阳性细胞率最高,依次为GDNF+GM1组、GDNF组、GM1组,组间比较差异有统计学意义(P＜0.05).单独应用GDNF和GM1不能诱导MSCs表达TH,二者联合应用可诱导MSCs表达TH,加入中脑条件培养基可使TH阳性细胞率明显增加,而且随着共培养时间的延长,TH阳性表达增加更显著.结论 中脑条件培养基与细胞因子联合可明显促进MSCs向神经元样细胞分化,并促进细胞表达TH.     

碱性成纤维细胞生长因子预诱导对骨髓基质干细胞向多巴胺能神经元分化的影响

目的探讨碱性成纤维细胞生长因子（bFGF）预诱导对骨髓基质干细胞（MSCs）向多巴胺（DA）能神经元分化的影响。方法取雄性Wistar大鼠股骨和胫骨骨髓，进行MSCs的体外培养、传代扩增及纯化。bFGF预诱导24h后，依据加入的神经营养因子不同分为单唾液酸四己糖神经节苷脂（GMl）组、胶质源性神经营养因子（GDNF）组和GDNF＋GMl组，以及对照组。倒置显微镜下观察细胞形态变化，分别在预诱导第3d、7d进行神经元特异性烯醇化酶（NSE）、神经胶质酸性蛋白（GFAP）、酪氨酸羟化酶（TH）免疫细胞化学检测。计数NSE和TH阳性细胞数，并计算阳性细胞百分比。结果对照组见少量NSE阳性细胞。实验组于诱导第3d、7d见较多数量的NSE、TH阳性细胞，GFAP阴性。bFGF预诱导各组中GDNF＋GMl组NSE、TH阳性细胞率最高，GDNF组次之，GMl组最低，组间比较差异有统计学意义（均P〈0．01）。结论bF—GF预诱导不仅可明显促进GDNF、GMl诱导MSCs向神经元样细胞分化，表达神经元细胞标志物——NSE；还可促进MSCs向DA能神经元分化，表达DA能神经元标志物——TH。     

Stem cell and precursor cell therapy

Strategies for cell replacement therapy have been guided by the success in the hematopoietic stem cell field. In this review, we discuss the basis of this success and examine whether this stem cell transplant model can be replicated in other systems where stem cell therapy is being evaluated. We conclude that identifying the most primitive stem cell and using it for transplant therapy may not be appropriate in all systems. We suggest alternative strategies such as progenitor cell replacement, inductive factors, bioengineering organs, in utero transplants, or any approach that takes advantage of the unique properties of the tissue and the stem cell type which, are more likely to provide effective functional replacement.     

Surface topography during neural stem cell differentiation regulates cell migration and cell morphology

We sought to determine the contribution of scaffold topography to the migration and morphology of neural stem cells by mimicking anatomical features of scaffolds found in vivo. We mimicked two types of central nervous system scaffolds encountered by neural stem cells during development in vitro by constructing different diameter electrospun polycaprolactone (PCL) fiber mats, a substrate that we have shown to be topographically similar to brain scaffolds. We compared the effects of large fibers (made to mimic blood vessel topography) with those of small‐diameter fibers (made to mimic radial glial process topography) on the migration and differentiation of neural stem cells. Neural stem cells showed differential migratory and morphological reactions with laminin in different topographical contexts. We demonstrate, for the first time, that neural stem cell biological responses to laminin are dependent on topographical context. Large‐fiber topography without laminin prevented cell migration, which was partially reversed by treatment with rock inhibitor. Cell morphology complexity assayed by fractal dimension was inhibited in nocodazole‐ and cytochalasin‐D–treated neural precursor cells in large‐fiber topography, but was not changed in small‐fiber topography with these inhibitors. These data indicate that cell morphology has different requirements on cytoskeletal proteins dependent on the topographical environment encountered by the cell. We propose that the physical structure of distinct scaffolds induces unique signaling cascades that regulate migration and morphology in embryonic neural precursor cells. J. Comp. Neurol. 524:3485–3502, 2016. © 2016 Wiley Periodicals, Inc.     

Schwann细胞及糖尿病性Schwann细胞病

周围神经病是糖尿病最常见的并发症[1],其发病机理历经探索,虽有诸多发现,但迄今尚未完全明了.本病以神经元数量减少,成鞘神经纤维和Schwann细胞变性为病理特征;因此,人们在研究病变神经元的同时,也十分关注其支持细胞即Schwann细胞,并提出"糖尿病性Schwann细胞病"的观点[2].几年来,有关Schwann细胞的生理病理研究广泛而深入,现综述如下:     

Stem cell reconstitution of autoimmune T cell repertoires

Maintenance of T cell memory in autoimmune disease may be complex because the unending renewable supply of self provides an inherent high antigen load that effectively precludes clearance, and because the broad array of potential immunogenic targets provides extensive self-recognition plasticity. Autoimmunity is characterized by a dynamic self-recognition process in which the primary autoreactivity initiating disease is soon followed and often displaced by secondary neoautoreactivities, or epitope spreading, that emerge as a result of endogenous self-priming. Here we show that the autoimmune disease process involves a tertiary phase of self recognition characterized by stem cell reconstitution of autoreactive T cells that recapitulates the myelin self recognition process involved in disease initiation and spreading during experimental autoimmune encephalomyelitis (EAE). Our study indicates that sustained autoimmune memory may not simply be due to the persistence of long-lived memory T cells, but may also involve bone marrow regeneration and replacement of the autoreactive T cell repertoire.     

Stem cell therapy for retinal ganglion cell degeneration

正The prospects of stem cell therapy for retinal ganglion cell(RGC)degeneration in human:RGC degeneration is a common pathologic cause of glaucoma and optic neuropathies,which are the leading cause of irreversible blindness and visual impairment in developed countries,currently affecting more than 100 million people worldwide.Intraocular pressure lowering can slow down glaucoma progression in a proportion of patients.Also,there is still no effective therapy for optic neuropathies.Besides,the degenerated RGCs in glaucoma cannot be     

Multiple cell types form the VIP amacrine cell population

Amacrine cells are a heterogeneous group of interneurons that form microcircuits with bipolar, amacrine and ganglion cells to process visual information in the inner retina. This study has characterized the morphology, neurochemistry and major cell types of a VIP-ires-Cre amacrine cell population. VIP-tdTomato and -Confetti (Brainbow2.1) mouse lines were generated by crossing a VIP-ires-Cre line with either a Cre-dependent tdTomato or Brainbow2.1 reporter line. Retinal sections and whole-mounts were evaluated by quantitative, immunohistochemical, and intracellular labeling approaches. The majority of tdTomato and Confetti fluorescent cell bodies were in the inner nuclear layer (INL) and a few cell bodies were in the ganglion cell layer (GCL). Fluorescent processes ramified in strata 1, 3, 4, and 5 of the inner plexiform layer (IPL). All tdTomato fluorescent cells expressed syntaxin 1A and GABA-immunoreactivity indicating they were amacrine cells. The average VIP-tdTomato fluorescent cell density in the INL and GCL was 535 and 24 cells/mm², respectively. TdTomato fluorescent cells in the INL and GCL contained VIP-immunoreactivity. The VIP-ires-Cre amacrine cell types were identified in VIP-Brainbow2.1 retinas or by intracellular labeling in VIP-tdTomato retinas. VIP-1 amacrine cells are bistratified, wide-field cells that ramify in strata 1, 4, and 5, VIP-2A and 2B amacrine cells are medium-field cells that mainly ramify in strata 3 and 4, and VIP-3 displaced amacrine cells are medium-field cells that ramify in strata 4 and 5 of the IPL. VIP-ires-Cre amacrine cells form a neuropeptide-expressing cell population with multiple cell types, which are likely to have distinct roles in visual processing.     

Granule cell number, cell death and cell proliferation in the dentate gyrus of wild-living rodents

Adult neurogenesis in the dentate gyrus occurs at species-specific levels. Wood mice (Apodemus flavicollis) show higher proliferation rates than laboratory mice and voles (Clethrionomys glareolus, Microtus subterraneus). We compare rates of cell death and proliferation and investigate if cell proliferation leads to the long-term recruitment of granule cells. Granule and pyknotic cell numbers were estimated in wild-living rodents in different age classes and compared with laboratory mice of mixed genetic background. All species differ significantly in their number of granule cells, except for the comparison of laboratory mice with European pine voles. Granule cell number is significantly higher in old bank voles and wood mice as compared to adults (23 and 37%, respectively). The number of pyknotic cells is highest in wood mice and lowest in laboratory mice. Across all species, the numbers of proliferating and pyknotic cells correlate. Despite differences in cell proliferation and cell death, the ratio of proliferating to pyknotic cells does not differ between adults of the wild-living species, but in laboratory mice a significantly lower proportion of cells die compared with the other species. In addition, the ratio of proliferating to pyknotic cells was significantly higher in old wood mice than in adults. We conclude (i) that cell proliferation can lead to an increase in granule cell number in wild-living rodents and (ii) that species- and age-specific changes of the ratio between proliferating and pyknotic cells occur as deviations from a close correlation of these two numbers across all species and age groups.     

Stem cell based cell therapy for muscular dystrophy]

Stem cell-based cell therapy is one of attractive therapeutic approaches to muscular dystrophy. In transplantation into dystrophic skeletal muscle, muscle satellite cells and musclE side population (SP) cells are good candidates as sources of stem cells. We purified mouse satellite cells from adult C57B16 mice by FACS using a monoclonal antibody, SM/C-2.6, which can specifically recognize quiescent satellite cells. DNA micro-array analysis on both quiescent and activated satellite cells allowed us to distinguish novel quiescent satellite cell-specific genes. These genes may encode molecules that keep satellite cells in a dormant and undifferentiated state. We transplanted purified muscle satellite cells transduced with lentivirus vector, and found these cells were effectively incorporated into dystrophin-deficient skeletal muscle and expressed transduced dystrophin. We also identified a novel side population (SP) cells in uninjured and regenerating skeletal muscle. The majority of muscle-SP cells in uninjured stage showed endothelial cell-like properties. CD31 negative/CD45 negative SP cells were a minor population in normal conditions, but actively proliferate during muscle regeneration. Co-transplantation experiments showed that the SP cells synergistically promoted muscle regeneration with satellite cells. It is indispensable to study molecular basis of muscle stem cells and muscle regeneration to achieve effective stem cell-based cell therapy on muscular dystrophy.     

TGF-beta induces cell death in the oligodendroglial cell line OLI-neu

We have shown that TGF-beta plays an important role during the period of developmental cell death in the nervous system. Immunoneutralization of TGF-beta prevents ontogenetic neuron death in vivo. Like neurons, oligodendrocytes are generated in excess and eliminated by apoptosis. It has been shown that oligodendrocyte progenitors and newly formed oligodendrocytes are especially susceptible to apoptosis. We choose the oligodendrocyte precursor cell line OLI-neu to address the question if TGF-beta could play a role for the control of oligodendrocyte proliferation and cell death. Flow cytometric analysis revealed that OLI-neu cells arrested in the G1 phase of the cell cycle underwent apoptosis in response to TGF-beta. TUNEL assays, apoptosis ELISA, and caspase assays substantiated the finding that OLI-neu cells died after TGF-beta treatment. Cell death could be inhibited by application of pan-caspase or caspase 8 and 9 inhibitors, whereas the inhibition of calpain was unaffected. Furthermore, we found a reduction of bcl-X(L) at the protein as well as at the mRNA level, while p27 was upregulated. The Smad cascade was activated while TGF-beta reduced the activity of the p42/p44 MAP kinase pathway. Together, these data show that TGF-beta induced apoptotic cell death in cells of oligodendroglial origin, whereby the signaling cascade involved the downregulation of antiapoptotic signaling such as bcl-X(L) leading to the activation of caspases.