大鼠脊髓匀浆上清诱导骨髓间充质干细胞分化为神经元样细胞的实验研究

目的:观察大鼠脊髓匀浆上清诱导骨髓间充质干细胞(mesenchymal stem cells,MSCs)形成的神经元样细胞形态特征.方法:通过贴壁法培养分离大鼠骨髓MSCs,体外扩增纯化后加入正常大鼠脊髓匀浆上清诱导72h,倒置显微镜下观察诱导前后细胞的形态结构.激光共聚焦显微镜观测钙离子细胞形态和荧光强度变化,免疫细胞化学方法鉴定诱导后细胞的表型特征.结果:倒置显微镜下可见MSCs呈纺锤形和多角形,核居中,有1-2个核仁,诱导后细胞呈神经元样,细胞伸出较长的轴突样和树突样突起.免疫细胞化学法显示NSE(神经元特异性烯醇化酶)、NF(神经丝蛋白)阳性,GFAP(神经胶质细胞酸性蛋白)阴性.共聚焦显微镜扫描脊髓匀浆上清诱导前细胞形态呈细长的梭形,细胞核不明显,胞体染色强,突起染色弱,荧光像素值低;诱导后,细胞呈现神经元样形态,胞体大,有多个突起,胞体及各突起染色强,荧光像素值高.结论:大鼠脊髓匀浆上清液可在体外诱导骨髓间充质干细胞分化为神经元样细胞.     

人胎儿脊髓神经干细胞的分离培养

本文旨在探讨是否能够从低温保存的流产儿分离培养出脊髓神经干细胞。将14周流产儿在4℃下保存,2、6和12h后取脊髓,将颈段、胸段、腰骶段分别进行无血清培养,并用胎牛血清诱导分化。用克隆培养的方法验证培养细胞的干细胞特性;用免疫荧光细胞化学的方法检测神经干细胞标志nestin及干细胞诱导分化后神经元标志MAP2、星形胶质细胞标志GFAP、胆碱能标志ChAT,并比较不同时间点以及不同部位分离的神经T细胞的差异。在各个时间点,从颈段、胸段、腰骶段脊髓均分离培养出具有连续增殖能力的神经球,其中腰骶段分离出的神经球数量最多,12h组各段分离出的神经球较2、6h组显著减少。各段培养中的神经球均为nestin阳性,诱导分化后均能够产生GFAP阳性星形胶质细胞、MAP2阳性神经元以及ChAT阳性胆碱能神经元。各段培养中的神经干细胞的克隆形成能力相似。以上结果表明,从低温保存的人胎儿能够分离培养出脊髓神经干细胞,这为基础研究以及未来治疗应用提供了新的细胞来源。     

神经上皮干细胞的分离培养及其体外分化特性的观察

目的探讨大鼠胚胎神经管神经上皮干细胞的分离培养条件,并观察其在体外的分化特性.方法采用显微解剖、机械吹打、无血清悬浮培养方法分离培养神经上皮干细胞,采用巢蛋白(nestin)免疫细胞化学染色技术检测神经上皮干细胞,用NSE和GFAP免疫组化染色检测并计数神经细胞和神经胶质细胞.结果大鼠胚胎神经管神经上皮干细胞在无血清培养基中可形成大量呈nestin抗原阳性细胞构成的神经球,经传代有血清培养后分化为NSE阳性和GFAP阳性细胞,其中NSE阳性细胞占细胞总数的47.7%,GFAP阳性细胞占细胞总数的39.8%.结论胎鼠神经管神经上皮干细胞在无血清培养中可增殖和传代,在有血清培养中可分化为神经细胞和神经胶质细胞,两者之比为47.7∶39.8.     

胎鼠脊髓神经干细胞分离方法的比较

目的：比较机械法和胰酶消化法对胎鼠脊髓源神经干细胞增殖分化的影响。方法：分别用机械法和胰酶消化法分离胎鼠脊髓组织获得神经干细胞,应用台盼蓝检测细胞成活率,用无血清培养技术培养神经干细胞,应用MTT法检测细胞分裂增殖能力．采用免疫细胞化学法鉴定神经干细胞和分化细胞。结果：机械法获得的细胞数量多于胰酶消化法。细胞经过培养其增殖能力机械法略强于胰酶消化法,但无统计学意义。培养形成的细胞球Nestin阳性,诱导分化后可见NSE和GFAP阳性细胞。结论：运用机械法比胰酶消化法分离胎鼠脊髓组织获得神经干细胞方法简单,容易操作,经过培养细胞增殖能力较强。并可提供健康的细胞来源。     

MiR-615在平行培养的神经干细胞与运动神经元的表达比较

目的探讨MiR-615在脊髓源性神经干细胞与运动神经元间的表达特征。方法通过免疫磁珠法分离纯化胚胎大鼠脊髓运动神经元;通过神经克隆球形成技术分离纯化胚胎大鼠脊髓源性神经干细胞。采用TaqMan miR-615Assay定量检测培养的脊髓源性神经干细胞与运动神经元中miR-615的表达差异。结果通过平行培养技术分别获得了纯化的脊髓源性神经干细胞与运动神经元。定量检测结果显示,miR-615在分离的运动神经元中较在脊髓源性神经干细胞中显著高表达。结论本文提示miR-615可能在神经干细胞定向分化为运动神经元过程中发挥重要的调节作用。     

胎鼠脊髓源性神经干细胞分离培养与鉴定

目的:研究胎鼠的脊髓源性神经干细胞的分离培养方法并观察其增殖和分化能力。方法:利用显微操作技术分离获得胎鼠脊髓组织、无血清培养技术和酶消化法结合机械法传代培养神经干细胞、免疫细胞化学方法鉴定神经干细胞和分化情况。结果:建立了胎鼠脊髓源性神经干细胞的分离、培养和鉴定的方法,观察到了脊髓源性神经干细胞具有较强的增殖能力,在添加有5ng／mlEGF和5ng／mlbFGF的无血清培养液中可贴壁分化为神经元、少突细胞和星形胶质细胞。结论:在体外培养条件下分离培养的胎鼠脊髓源性神经干细胞具有干细胞的特性即较强的增殖能力和多向分化潜能。     

新生鼠中枢神经系统内Nestin蛋白免疫阳性的组织分布

为了观察Nestin在新生SD大鼠中枢神经系统中的分布,探讨神经干细胞在新生鼠的分布.采用免疫荧光法,显示含神经干细胞特征性的标志物Nestin的阳性结构在新生SD大鼠中枢神经系统中的分布.结果表明在新生SD大鼠中枢神经系统中,Nestin在前脑、脑干和小脑的各个部位均有表达,阳性结构多为纤细的纤维状突起,分布密集,标记强度多为中等强度,分布相对比较均匀.在脊髓实质的Nestin免疫阳性产物明显减少,分布稀疏,染色也较浅,中央管Nestin免疫染色阳性的室管膜细胞很少,但在脊髓中央管的背侧(延髓见于腹侧和背侧)可见到“喷泉”状免疫强阳性纤维束垂直伸展,直达软膜.由此可得出结论:新生SD大鼠中枢神经系统的广泛脑区均存在大量的神经干细胞,而脊髓的神经干细胞数目较少,提示神经干细胞在生后从神经系统的尾端开始逐渐减少.     

胚胎大鼠中枢神经系统神经干细胞分离培养方法研究

为了建立经济实用的神经干细胞培养方法 ,以Wistar大鼠胚龄 1 4～ 1 5天的胚胎为实验材料 ,选取中枢神经系统的大脑和小脑神经组织 ,经 0 1 2 5 %胰酶消化处理 ,在生长因子 (aFGF和bFGF)作用下 ,采用无血清培养法进行培养。通过倒置相差显微镜进行观察 ,可观察到明显的干细胞的不对称分裂图像。神经干细胞在培养一段时间后均出现细胞团 ,细胞团贴壁后可进行分化 ,形态上表现为神经元样和神经胶质样细胞。利用激光扫描共聚焦显微镜对其进行鉴定 ,可检测到神经干细胞标志性蛋白-Nestin蛋白。采用此法可以分离得到具有干细胞特征和多分化潜能的神经干细胞 ,建立了一套神经干细胞的分离、培养和鉴定的方法 ,这将对更好地理解神经系统的发育机制 ,及临床治疗神经系统疾病具有重要意义。     

麝香水溶物对大鼠神经干细胞的生长、分化和电转染率的影响

目的：研究麝香水溶物对培养的大鼠神经干细胞的生长、分化和电转染率的影响。方法：在培养基中加入不同浓度的麝香水溶性提取物后,观察大鼠神经干细胞的生长分化情况;利用表达增强型绿色荧光蛋白的质粒pEGFP-C1,对麝香水溶物处理的神经干细胞进行电穿孔转染,调查电转染率。结果：麝香水溶物处理后的大鼠神经干细胞的细胞团分散,神经突起增多、变长,贴壁细胞增加,细胞形态呈多样性。在0.3‰浓度下,神经干细胞有向神经胶质样细胞分化的趋势。对于麝香处理后变化的细胞,再转到正常培养基中后,细胞基本都能恢复到正常的神经干细胞形态,浓度较高（3‰）时,细胞的恢复能力下降,部分细胞因细胞膜受损严重而死亡。电转染结果表明,麝香处理后发生变化的细胞对pEGFP-C1的电转染率明显提高。结论：麝香水溶物能促进大鼠神经干细胞团的分散和细胞贴壁、变形,并有向神经胶质样细胞分化的趋势。同时可以提高神经干细胞对pEGFP-C1的电转染率。     

大鼠胚胎神经干细胞单克隆化及单层化培养和鉴定

采用原代培养SD胎鼠神经干细胞,在形成神经球之后,传代至0.1%明胶包被的培养皿,显微镜下挑取一个神经球贴壁后的细胞团,吹打后贴壁培养．同样方法挑细胞团并传代培养5～6次,得到纯化的由一个神经干细胞扩增的克隆,对得到的神经干细胞进行鉴定以及分化能力的评估,证明得到的细胞就是神经干细胞．结果表明,成功分离了SD胎鼠的神经干细胞,进行单克隆化单层培养,神经干细胞和分化后的细胞标志基因都可以检测到．上述工作为疾病模型大鼠治疗及相关基础研究提供细胞来源及形态标准．     

Neural progenitors isolated from newborn rat spinal cords differentiate into neurons and astroglia

Permanent functional deficit in patients with spinal cord injury (SCI) is in part due to severe neural cell death. Therefore, cell replacement using stem cells and neural progenitors that give rise to neurons and glia is thought to be a potent strategy to promote tissue repair after SCI. Many studies have shown that stem cells and neural progenitors can be isolated from embryonic, postnatal and adult spinal cords. Recently, we isolated neural progenitors from newborn rat spinal cords. In general, the neural progenitors grew as spheres in culture, and showed immunoreactivity to a neural progenitor cellular marker, nestin. They were found to proliferate and differentiate into glial fibrillary acidic protein-positive astroglia and multiple neuronal populations, including GABAergic and cholinergic neurons. Neurotrophin 3 and neurotrophin 4 enhanced the differentiation of neural progenitors into neurons. Furthermore, the neural progenitors that were transplanted into contusive spinal cords were found to survive and have migrated in the spinal cord rostrally and caudally over 8 mm to the lesion center 7 days after injury. Thus, the neural progenitors isolated from newborn rat spinal cords in combination with neurotrophic factors may provide a tool for cell therapy in SCI patients.     

Reaggregates of cells from rat testis resemble developing gonads

Reaggregates prepared from newborn rat testis cells in Moscona-type rotation cultures were analyzed and compared with normal fetal (12-21 days) and newborn testes at the light and electron microscope level. After 25 h of culture, the aggregates resembled normal testicular tissue. The cells of the surface layer were spindle-shaped and connected by adherent junctions. The epithelial cords were composed exclusively of Sertoli cells and were surrounded by elongated cells resembling the developing myoid cells in newborn testes. The basal aspect of the cords was covered by a layer of flocculent material which, in places, was organized like an ordinary basement membrane. Individual spermatogonia with pseudopodes were observed in the interstitial tissue. Some Leydig cells were organized into small clusters like those typical in newborn testes. The present observations indicate that, histologically, the reaggregation of separated testicular cells resembles the differentiation of embryonic male gonads.     

骨髓间充质干细胞移植修复大鼠半横断脊髓损伤

目的初步探讨骨髓间充质干细胞诱导为神经细胞,及其移植对大鼠脊髓半横断损伤神经功能恢复和运动的影响。方法贴壁培养法分离培养大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs),大鼠脊髓匀浆上清诱导第3代向神经细胞分化,经免疫组化鉴定分化后细胞的性质。制备大鼠半横断脊髓损伤模型,脊髓损伤局部注射BrdU标记诱导后的神经细胞。细胞移植5周后观察移植细胞在脊髓内存活分布情况。结果倒置显微镜下可见MSCs呈纺锤形和多角形,有1~2个核仁,经脊髓匀浆上清诱导后,发出数个细长突起,并交织成网,诱导后的细胞表达Nestin,可推测诱导后的细胞为MSCs源神经细胞。5周后移植的MSCs在宿主损伤脊髓内聚集并存活,表达MAP-2、NF、GFAP与对照组比较有统计学意义(P0.05)。大鼠运动功能较移植前有所改善。结论MSCs经脊髓匀浆上清诱导后移植治疗大鼠半横断脊髓损伤可使运动功能得到改善。     

Biosynthesis of NAAG by an enzyme-mediated process in rat central nervous system neurons and glia

The peptide transmitter N-acetylaspartylglutamate (NAAG) is present in millimolar concentrations in mammalian spinal cord. Data from the rat peripheral nervous system suggest that this peptide is synthesized enzymatically, a process that would be unique for mammalian neuropeptides. To test this hypothesis in the mammalian CNS, rat spinal cords were acutely isolated and used to study the incorporation of radiolabeled amino acids into NAAG. Consistent with the action of a NAAG synthetase, inhibition of protein synthesis did not affect radiolabel incorporation into NAAG. Depolarization of spinal cords stimulated incorporation of radiolabel. Biosynthesis of NAAG by cortical astrocytes in cell culture was demonstrated by tracing incorporation of [3H]-glutamate by astrocytes. In the first test of the hypothesis that NAA is an immediate precursor in NAAG biosynthesis, [3H]-NAA was incorporated into NAAG by isolated spinal cords and by cell cultures of cortical astrocytes. Data from cerebellar neurons and glia in primary culture confirmed the predominance of neuronal synthesis and glial uptake of NAA, leading to the hypothesis that while neurons synthesize NAA for NAAG biosynthesis, glia may take it up from the extracellular space. However, cortical astrocytes in serum-free low-density cell culture incorporated [3H]-aspartate into NAAG, a result indicating that under some conditions these cells may also synthesize NAA. Pre-incubation of isolated spinal cords and cultures of rat cortical astrocytes with unlabeled NAA increased [3H]-glutamate incorporation into NAAG. In contrast, [3H]-glutamine incorporation in spinal cord was not stimulated by unlabeled NAA. These results are consistent with the glutamate-glutamine cycle greatly favoring uptake of glutamine into neurons and glutamate by glia and suggest that NAA availability may be rate-limiting in the synthesis of NAAG by glia under some conditions.     

Primary culture of axolotl spinal cord ependymal cells

In order to examine the role of ependymal cells in the spinal cord regeneration of urodele amphibians, procedures were established to identify and culture these cells. Cell isolation and culture conditions were determined for ependymal cells from larval and adult axolotls (Ambystoma mexicanum). Dissociated cells prepared from intact spinal cords were cultured on fibronectin- or laminin-coated dishes. Dissociated cells attached more rapidly to fibronectin, but attached and spread on both fibronectin and laminin. Essentially pure populations of ependymal cells were obtained by removing 2 week old ependymal outgrowth from lesion sites of adult spinal cords. These ependymal outgrowths attached and grew only on fibronectin-coated dishes. Growth and trophic factors were tested to formulate a medium that would support ependymal cell proliferation. The necessary peptide hormones were PDGF, EGF, and insulin. TGF-beta(1) affected the organization of cell outgrowth. Initially, longterm culture required the presence of high levels of axolotl serum. Addition of purified bovine hemaglobin in the culture medium reduced the serum requirement. Outgrowth from expiants was subcultured by transferring groups of cells. Intrinsic markers were used to identify ependymal cells in culture. The ependymal cells have characteristic ring-shaped nucleoli in both intact axolotl spinal cords and in culture. Indirect immunofluorescence examination of intermediate filaments showed that ependymal cells were glial fibrillary acidic protein (GFAP) negative and vimentin positive in culture. Identification of dividing cells was made using (3)H-thymidine incorporation and autoradiography, and by the presence of mitotic figures in the cultured cells.     

Sox11 promotes endogenous neurogenesis and locomotor recovery in mice spinal cord injury

We introduced a lentiviral vector containing the Sox11 gene into injured spinal cords of mice to evaluate the therapeutic potential of Sox11 in spinal cord injury. Sox11 markedly improved locomotor recovery after spinal cord injury and this recovery was accompanied by an up-regulation of Nestin/Doublecortin expression in the injured spinal cord. Sox11 was mainly located in endogenous neural stem cells lining the central canal and in newly-generated neurons in the spinal cord. In addition, Sox 11 significantly induced expressions of BDNF in the spinal cords of LV-Sox11-treated mice. We concluded that Sox11 induced activation of endogenous neural stem cells into neuronal determination and migration within the injured spinal cord. The resultant increase of BDNF at the injured site might form a distinct neurogenic niche which induces a final neuronal differentiation of these neural stem cells. Enhancing Sox11 expression to induce neurogenic differentiation of endogenous neural stem cells after injury may be a promising strategy in restorative therapy after SCI in mammals.     

Nogo—p4与NgR结合抑制神经干细胞分化为双极形星形胶质细胞的突起生长

目的观察Nogo—p4是否通过与NgR结合的途径对大鼠脊髓来源神经干细胞分化形成双极形星形胶质细胞突起长度产生抑制。方法取4只出生24h内的Wistar大鼠,悬浮培养法培养大鼠脊髓来源的神经干细胞。把神经干细胞分为A、B、C、D四组,A组加入血清,B组加入血清和Nogo—p4,C组神经干细胞经RNA干扰沉默NgR基因后加入血清分化,D组神经干细胞经RNA干扰沉默NgR基因后加入血清和Nogo—p4。分化第7d,GFAP抗体标记星形胶质细胞,使用Image—ProPlus5．0软件测量双极形星形胶质细胞突起长度。结果神经干细胞分化第7d,四组均可形成双极形星形胶质细胞。B组中双极形星形胶质细胞的突起长度明显短于其它各组。A、C、D组中双极形星形胶质细胞的突起长度没有显著差异。结论Nogo—p4经与NgR结合途径显著抑制脊髓来源神经干细胞分化成的双极形星形胶质细胞的突起生长。