基质细胞衍生因子-1诱导神经干细胞靶向性迁移的实验研究

目的 观察基质细胞衍生因子-1(SDF-1)对神经干细胞(NSCs)迁移的影响.方法 由胚胎大鼠脑组织获取NSCs并进行传代培养和分化鉴定,使用流式细胞术检测NSCs纯度及SDF-1特异性受体CXCR4的表达情况,之后利用Blind-Well小室体外迁移体系观察不同浓度的SDF-1(0 ng/L、1 ng/mL、10 ng/mL、50 ng/mL、100 ng/mL、500 ng/mL和1000 ng/mL)对NSCs迁移数量的影响,并使用μ-载片观察SDF-1对NSCs迁移距离的影响.结果成功分离培养得到了能够在体外不断分裂增殖、具有多向分化潜能的NSCs,连续传3代后绝大部分细胞nestin表达阳性,nestin和CXCR4的共表达率达到80%左右.细胞趋化实验结果表明,SDF-1对NSCs有较强的趋化作用,随着SDF-1浓度的升高,发生迁移的细胞数量也随之增加,并于SDF-1浓度为500ng/mL时达到最高峰[(256.79±38.27)个细胞/每高倍视野].在μ-载片细胞生长通道两侧的SDF-1浓度梯度(500ng/mL→0ng/mL)作用下,由细胞克隆球迁出的细胞呈不对称分布,通常有更多的迁出细胞分布于趋化因子浓度较高的一侧,且该侧细胞的最大迁移距离也比对侧远.结论 SDF-1与其特异性受体CXCR4相巨作用能够诱导NSCs发生靶向性迁移.     

神经干细胞原代培养方法的优化

目的 探讨低浓度胰酶不同消化分离时间对体外培养新生大鼠海马NSCs增殖与凋亡的影响.方法 取出生24 h内SD大鼠海马组织,以1.25 g/L胰酶37℃分别消化5、10、15、20和25min(依次为A～E组),获得单细胞悬液后进行培养.通过台盼蓝染色计数、细胞形态观察和神经球数目比较不同消化时间对NSCs活力和生长的影响;用5-溴-2脱氧尿嘧啶核苷(BrdU)标记法检测NSCs的增殖能力;用免疫荧光细胞化学法检测BrdU、nestin的表达:用Annexin V-FITC/PI染色和流式细胞仪检测细胞凋亡率.结果 原代和传代培养的NSCs都能快速增殖并形成神经球;免疫荧光染色结果显示神经球细胞均表达NSCs特异性标志物nestin;所获得的细胞能将BrdU结合到细胞核中;各组培养3、5、7 d后,C组(消化15 min)NSCs成球数最多,BrdU标记克隆率最高,细胞凋亡率最低,与其他组比较差异有统计学意义(P<0.05).结论 体外分离培养的新生大鼠海马NSCs具有增殖能力,1.25 g/L胰酶在不同消化时间对NSCs增殖能力和凋亡率的影响有所不同,消化时间过长或过短都会抑制NSCs增殖,诱导NSCs凋亡,且消化时间越长NSCs的凋亡率越高.

Abstract：

Objective To study the influence of digestion times of low concentration trypsin on the proliferation and apoptosis of neural stem cells (NSCs) in the hippocampus of neonate rats.Methods Hippocampus of neonatal rats (within 24 h) were taken out, and treated with trypsin at 1.25g/L concentration and 37 ℃ for 5, 10, 15, 20 and 25 minutes; unicellular suspension was then successfully got and primary culture and subculture were performed. Effects of trypsinization on cell viability and growth of NSCs were compared by observing the cell morphology and Trypan blue staining.The 5-bromodeoxyuridine labeling was performed to assess the self-renewing and proliferative activities of NSCs. Fluorescence immunocytochemistry was carried out to examine the expressions of BrdU and nestin. Apoptosis was measured by Annexin V-FITC/PI assay and flow cytometry. Results Primary and passage culture of NSCs enjoyed rapid proliferation and formation of neurospheres. The neurosphere cells expressed NSCs specific marker nestin by immunofluorescence; all the neurosphere cells could incorporate BrdU into the nucleus; of the neurospheres obtained from the 3rd, 5th and 7th d, those digested for 15 rain enjoyed the highest level of NSCs neurospheres, the highest BrdU labeled clone and the lowest cell apoptosis as compared with those digested for 5, 10, 20 and 25 min (P<0.05). Conclusion The NSCs isolated from the hippocampus of neonatal rats have the ability of proliferation in vitro. And 1.25 g/L concentration of trypsin with digestion times could positively change the proliferative and apoptosis capacity of NSCs: too short or long digestion times can inhibit the proliferation of NSCs and induce the apoptosis of NSCs; the longer the digestion time, the higher the apoptosis of NSCs.     

嗅鞘细胞对原代培养神经干细胞增殖、分化的影响

目的 观察嗅鞘细胞(OECs)对神经干细胞(NSCs)增殖、分化的影响.方法 新生大鼠脑OECs和NSCs原代培养,采用免疫荧光及免疫细胞化学方法鉴定相关细胞.取原代OECs分为2组,实验组去除培养孔的间隔,使OECs和NSCs共用一培养液体系;对照组不破坏培养孔的间隔,单独培养NSCs,其余同实验组.观察2组细胞增殖、分化情况.结果 原代培养的OECs表达神经生长因子受体(P75NGFR);原代培养的神经球表达巢蛋白(nestin),神经球分化的细胞表达神经丝200(NF200)和胶质纤维酸性蛋白(GFAP).增殖实验中,实验组NSCs数量较对照组明显增多,差异有统计学意义(P<0.05).诱导分化实验中,实验组4d、7d时NF200阳性细胞率较对照组明显升高,差异有统计学意义(P<0.05),说明2种细胞共液培养时,OECs提高了NSCs向NF200阳性细胞分化率.结论 OECs可促进NSCs增殖,并提高了NSCs向神经元分化的效率.     

胚胎大鼠嗅神经干细胞的培养及分化特性

目的建立胚胎大鼠嗅神经干细胞(NSCs)体外培养方法,研究其增殖和分化特性.方法采用添加丝裂原的无血清培养基分离、培养胚胎14 d(E14)大鼠嗅球NSCs,应用免疫细胞化学方法鉴定培养的NSCs及自然分化为特异性神经细胞的类型,测定NSCs的生长曲线.结果从E14大鼠嗅球分离、培养出表达nestin,并能分化为神经元、星形胶质细胞和少突胶质细胞的NSCs.嗅NSCs的增殖依赖表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF),其中EGF的促分裂增殖作用明显优于bFGF.结论从E14大鼠嗅球培养出具有自我增殖和多向分化潜能的NSCs.     

Neural stem cell, as a source of graft material for transplantation in neuronal disease

Self-renewing and multipotent neural stem cells are present in the adult human brain. We successfully harvested neural stem cells from mice and humans using misexpressed EGFP proteins under the control of the nestin second intron enhancer. High-level EGFP expressors derived from mouse embryos included a distinct subpopulation of cells that were self-renewable and multipotent. Further, we obtained that neural progenitor cells from rat fetal spinal cords using a neurosphere technique, and demonstrated their ability to divide and differentiate into neurons in vivo, where they were integrated into the host tissue in the injured rat spinal cord with resultant behavioral improvement of the recipient rat. We also harvested tyrosine hydroxylase-positive neurons from a transgenic mouse expressing GFP under the control of the tyrosine hydroxylase promoter, and successfully transplanted them into the striatum of rats with parkinsonism with marked improvement of the neurological symptoms. Since neural stem cells can adapt well in the host CNS, studies should focus on their application as a vector in gene therapy and on the introduction in vivo or ex vivo of genes to control their proliferation and differentiation. Neural stem cells are a potential, useful source for developing new therapy for CNS disorders.     

胚胎小鼠脊髓源性与纹状体源性神经干细胞的培养及分化特点

目的 探讨小鼠脊髓源性神经干细胞与纹状体源性神经干细胞的分离培养方法 及增殖特点,比较两种来源的神经干细胞发育时期上的异同,寻找更有利于脊髓损伤修复的种子细胞.方法 利用显微解剖、无血清培养和单细胞克隆技术在孕14 d小鼠的胎鼠的脊髓及纹状体中分离培养具有单细胞克隆能力的细胞,免疫荧光染色检测克隆细胞的神经巢蛋白(nestin)抗原和诱导分化后特异性成熟神经细胞抗原的表达,并比较两种来源的干细胞在培养及分化方向上的异同点.结果从胎鼠的脊髓和纹状体中成功分离出神经干细胞.两种来源的干细胞均具有连续克隆能力可传代培养,表达nestin.脊髓血清诱导分化后脊髓源性神经干细胞β-tubulinⅢ阳性细胞(13.5±0.8)较纹状体源性神经干细胞(17.4±1.1)减少,而nestin、GFAP阳性细胞明显增多(45.7±0.3vs 39.2±1.2;25.2±1.3 vs 18.8±0.9),差异均有统计学意义(P<0.05). 结论 依据细胞增殖特点和分化结果的区别,证实纹状体源性神经干细胞更适合用于移植修复脊髓损伤.     

Nestin enhancer requirements for expression in normal and injured adult CNS

The nestin gene is expressed in many CNS stem/progenitor cells, both in the embryo and the adult, and nestin is used commonly as a marker for these cells. In this report we analyze nestin enhancer requirements in the adult CNS, using transgenic mice carrying reporter genes linked to three different nestin enhancer constructs: the genomic rat nestin gene and 5 kb of upstream nestin sequence (NesPlacZ/3), 636 bp of the rat nestin second intron (E/nestin:EGFP), and a corresponding 714 bp region from the human second intron (Nes714tk/lacZ). NesPlacZ/3 and E/nestin:EGFP mice showed reporter gene expression in stem cell-containing regions of brain and spinal cord during normal conditions. NesPlacZ/3 and E/nestin:EGFP mice showed increased expression in spinal cord after injury and NesPlacZ/3 mice displayed elevated expression in the periventricular area of the brain after injury, which was not the case for the E/nestin:EGFP mice. In contrast, no expression in adult CNS in vivo was seen in the Nes714tk/lacZ mice carrying the human enhancer, neither during normal conditions nor after injury. The Nes714 tk/lacZ mice, however, expressed the reporter gene in reactive astrocytes and CNS stem cells cultured ex vivo. Collectively, this suggests a species difference for the nestin enhancer function in adult CNS and that elements outside the second intron enhancer are required for the full injury response in vivo.     

Prospective characterization of neural stem cells by flow cytometry analysis using a combination of surface markers

Neural stem cells (NSCs) with self-renewal and multilineage differentiation properties can potentially repair degenerating or damaged neural tissue. Here, we have enriched NSCs from neurospheres, which make up a heterogeneous population, by fluorescence-activated cell sorting (FACS) with antibodies against syndecan-1, Notch-1, and integrin-beta1, which were chosen as candidates for hematopoietic cell-or somatic stem cell-markers. Antigen-positive cells readily initiated neurosphere formation, but cells lacking these markers did so less readily. Doubly positive cells expressing both syndecan-1 and Notch-1 underwent neurosphere formation more efficiently than did singly positive cells. The progeny of sorted cells could differentiate into neurons and glial cells both in vitro and in vivo. These antibodies were also useful for isolating cells from the murine embryonic day 14.5 brain that efficiently formed neurospheres. In contrast, there was no distinct difference in neurosphere formation efficiency between Hoechst 33342-stained side population cells and main population cells, although the former are known to have a stem cell phenotype in various tissues. These results indicate the usefulness of syndecan-1, Notch-1, and integrin-beta1 as NSC markers.     

不同方法诱导大鼠脂肪基质细胞定向分化为神经细胞的比较研究

目的 探索提高大鼠脂肪基质细胞(ADSCs)向神经细胞定向诱导分化比例的实验方法.方法 根据所采用的不同细胞诱导分化方法分为A组(直接诱导组:以含血清及神经营养因子的Neurobasal培养基直接诱导ADSCs向神经细胞分化)和B组[神经球诱导组:先以无血清Neurobasal培养基将ADSCs诱导为神经干细胞(NSCs)球,再以含血清及营养因子的Neurobasal培养基诱导神经球干细胞向神经细胞分化].通过细胞形态学观察以及免疫细胞化学检测巢蛋白(nestin)、β-微管蛋白Ⅲ(β-tubulinⅢ)、微管相关蛋白2(MAP2ab)和胶质纤维酸性蛋白(GFAP)表达,鉴定细胞分化结果.结果 免疫细胞化学结果证实,两组细胞均有效表达nestin和β-tubulinⅢ.A组细胞在诱导6 h时nestin表达可达高峰(73.8%±6.5%),并很快下降(24 h为50.3%±3.8%,72 h为10.5%±3.0%),72 h后即检测不到;β-tubulinⅢ在诱导后24 h开始表达(33.5%±6.6%),1周达高峰(84.3%±33%).B组nestin的高表达持续整个神经球阶段,β-tubulinⅢ在神经球阶段有少量表达(成球后7 d为14.1%±3.3%),神经球细胞分化后其表达明显增加(分化后3 d为46.4%±6.1%);B组存由NSCs向神经细胞诱导一定时间后可检测到一定比例的MAP2ab阳性细胞(24.5%).结论 ADSCs在体外稳定扩增传代并在适宜诱导条件下,可以向神经细胞定向分化.应用神经球诱导法和直接诱导法均可得到较高比例的nestin及β-tubulinⅢ阳性细胞,神经球诱导组nestin表达稳定,并有一定比例MAP2ab阳性细胞出现.     

Wnt3a影响神经干细胞分化的体外研究

目的探讨Wnt3a对胚胎大鼠海马神经干细胞（NSCs）体外分化的影响。方法采用机械分离、无血清传代培养法从胎鼠海马中获得NSCs,使用免疫荧光法对其干细胞特性及其受体Fzd3蛋白表达进行鉴定,观察Wnt3a对NSCs体外分化的影响。结果海马NSCs表达特异性标志物巢蛋白及胞膜蛋白Fzd3;体外诱导分化,Wnt3a处理组神经元及星形胶质细胞分化的比例分别为11．25％±0．62％和56．26％±4．82％,而对照组则为8．54％±0．48％和168．42％±5．54％;组间分化差异具有统计学意义（P〈0．05）。结论体外环境下Wnt3a能够促进NSCs向神经元分化,并抑制其向星形胶质细胞分化。     

神经干细胞在wistar大鼠体内、外追踪胶质瘤细胞的特性

目的 观察在胶质瘤细胞存在这一病理状态下,神经干细胞(neural stem cells,NSCs)在体内、外的迁移特点;了解导入NSCs的外源基因在迁移过程中的表达情况。方法 分离、培养NSCs,脂质体介导pIRES2-EGFP质粒转染NSCs,BrdU标记NSCs;共同培养胶质瘤细胞和NSCs,观察NSCs在体外的迁移特点;制作大鼠颅内胶质瘤模型,用转染EGFP基因和BrdU标记的NSCs接种颅内胶质瘤,免疫组织化学检测,观察NSCs在体内的迁移特点,了解导入NSCs的外源基因在颅内的表达情况。结果 培养成功了Wistar胎鼠大脑NSCs,成功将EGFP基因导入NSCs,NSCs在体内、外显示了对胶质瘤细胞强烈的追踪特性,同时表达外源基因的产物。结论 NSCs在体内、外具有追踪胶质瘤细胞的特性,同时能够表达外源基因的产物。     

神经干细胞移植治疗大鼠脑缺血再灌注损伤实验研究

目的探讨大鼠胚胎神经干细胞移植治疗局灶性脑缺血再灌注损伤的可行性。方法孕龄8~10d的大鼠神经干细胞在体外扩增后,用免疫组织化学方法分别检测神经干细胞及其分化后代的特异性标志蛋白nestin、胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)的表达。分别于缺血后不同时间窗将神经干细胞移植到局灶性脑缺血大鼠模型的缺血半暗带和梗塞中心,移植4w后比较不同移植部位神经干细胞存活、增殖和迁移的差异。结果从胎鼠中成功培养出悬浮生长的可表达nestin的神经球,其在含血清条件下可分化为表达GFAP的胶质细胞和表达NSE的神经元。神经干细胞移植4w后可见所有移植动物的细胞都存活,梗塞中心移植的细胞存活、增殖水平明显低于半暗带移植的细胞。结论大鼠胚胎神经干细胞移植到局灶性脑缺血再灌注损伤大鼠梗塞中心和半暗带均可长期存活,其增殖能力与移植部位密切相关。     

神经干细胞移植治疗大鼠创伤性脑外伤的实验研究

目的 将神经干细胞(NSCs)移植于大鼠创伤性脑损伤部位,观察NSCs对创伤性脑损伤的治疗作用.方法 首先进行NSCs的体外培养,同时采用5-2-溴脱氧尿嘧啶(BrdU)标记,脑外伤模型采用自由落体撞击大鼠左侧大脑皮质运动感觉区来制作,脑外伤后24h内移植NSCs,分别于脑外伤前、脑外伤后1、2、3周时行神经运动行为学评分(NMFE)和免疫组织化学染色检测BrdU、巢蛋白(nestin)、神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)和半乳糖脑苷酯(GalC)蛋白表达和TUNEL原位杂交染色,经计算机图像分析系统处理.结果 大鼠自由落体撞击伤后,右下肢神经运动功能障碍明显.神经运动行为学评分在第1周时,实验组与对照组无明显差异,而在第2、3周时,实验组的评分明显低于对照组,有统计学意义(P＜0.05).第2、3周时实验组NSE、GFAP和GalC染色阳性细胞数要多于对照组,nestin染色在第1周时表达最高,而后逐渐下降.BrdU阳性细胞在损伤灶中心区最多,在损伤灶的远隔部位也有少许发现,而TUNEL染色则对照组明显比NSCs组高,有统计学意义(P＜0.05).结论 脑外伤移植NSCs可以在损伤区存活、增殖及分化,并在损伤的远隔部位有少许NSCs迁移、分化.NSCs移植有利于大鼠脑自由落体撞击伤后期的功能恢复.     

多聚赖氨酸和明胶对神经干细胞增殖和分化的影响

摘要 背景：目前大鼠神经干细胞体外诱导分化的研究报道诸多,但其分化过程很难控制,很多实验的操作方法复杂,分化比率也很低。 目的：探索大鼠胚胎前脑神经干细胞体外原代及传代培养方法,并观察其分化规律。 方法：胎鼠在无菌条件下分离出前脑,制备单细胞悬液,以1×1011L-1接种于含N2的DMEM/F12培养基中培养,传代培养过程中加入BrdU,标记神经干细胞球。诱导分化实验分为多聚赖氨酸铺板组、明胶铺板组和无铺板组。采用体积分数20%胎牛血清刺激其分化。免疫细胞化学检测nestin、BrdU及在血清诱导条件下神经干细胞向神经细胞分化的能力。 结果与结论：细胞呈神经干细胞样生长,具有连续增殖能力,可以传代培养。传代神经球中的细胞均呈nestin阳性和BrdU阳性。多聚赖氨酸铺板组和明胶铺板组贴壁后分化为神经细胞能力强于无铺板组(P < 0.01)。多聚赖氨酸铺板组略强于明胶铺板组(P > 0.05)。神经谱系标记物神经胶质纤维酸性蛋白和微管相关蛋白2的免疫细胞化学结果均阳性。结果表明,大鼠胚胎前脑富含神经干细胞,其分化观察,多聚赖氨酸和明胶在诱导神经干细胞分化中作为细胞贴壁支持物提高分化细胞数量的作用,且多分化为星形胶质细胞。 关键词：多聚赖氨酸;神经干细胞;明胶;增殖分化;体外培养 doi:10.3969/j.issn.1673-8225.2010.47.004     

小鼠神经干细胞的生物学特性观察

目的探讨小鼠神经干细胞体外原代培养的生长特性.方法用无血清与单细胞克隆技术对小鼠胚胎脑组织进行分离、培养,用光镜、免疫组织化学进行鉴定,并对其不同生长时期的生物学特性进行透射电镜观察.结果鼠胚胎分离细胞具有连续分化及克隆能力,克隆球和早期的原始细胞神经上皮干细胞蛋白(nestin)抗原呈阳性.成熟分化后胶质纤维酸性蛋白(GFAP)及神经丝-200(NF-200)抗原呈阳性.不同期电镜结果:培养2周的神经干细胞较原始,核大,细胞质少,细胞器不发达.将其继续培养到4周,部分细胞内可见到发达的细胞器,并在分化细胞中观察到神经微管、微丝、胶样丝和细胞间连接样结构.结论胚胎脑组织在体外培养并克隆成神经球,具有很强的增殖能力,是多分化潜能干细胞.     

LIF receptor signaling modulates neural stem cell renewal

Activation of the leukemia inhibitory factor (LIF) receptor has been reported to promote gliogenesis and also to support neural stem cell (NSC) renewal. To investigate this paradox, we isolated NSCs and generated neurospheres from embryonic mice either wild-type, heterozygous, or homozygous null for LIF receptor (LIFR)-beta. Exogenous LIF abrogated neurosphere formation and promoted expression of GFAP by all cells in wild-type and heterozygous cultures. LIF also stimulated a twofold increase in the number of multipotential clones generated from these cultures in comparison with those pretreated with EGF and FGF-2 (E+F) alone. In contrast, the clonogenicity of low-density cultures of LIFR knockout cells was reduced in comparison with that of wild-type cells grown in E+F and was unaffected by LIF. Thus, although LIFR signaling is not necessary for NSC self-renewal, it enhances both the clonogenicity and the expression of GFAP by these multipotential cells.     

Developmental expression of glial fibrillary acidic protein mRNA in mouse forebrain germinal zones--implications for stem cell biology

Postnatal neural stem cells (NSCs) express the "traditional" astrocyte marker, glial fibrillary acidic protein (GFAP). Here, we analyze the ontogeny of GFAP mRNA in mouse forebrain germinal zones (GZ). On embryonic day 15, mRNA distribution is highly restricted. Subsequently, expression expands to include many cells in the GZ regions adjacent to the cortex and septum but not to the striatum. Double immunostaining for GFAP and nestin did not demonstrate extensive overlap in the GZ of adult rats, suggesting that either few of the GFAP-expressing cells are stem cells, or that nestin is not a reliable marker for stem cells in the adult rat brain. The current findings indicate that while some GFAP-expressing cells in the GZ may be NSCs, most are not likely to function in a neurogenic capacity.     

Phenotypic and functional heterogeneity of GFAP-expressing cells in vitro: differential expression of LeX/CD15 by GFAP-expressing multipotent neural stem cells and non-neurogenic astrocytes

Recent findings show that the predominant multipotent neural stem cells (NSCs) isolated from postnatal and adult mouse brain express glial fibrillary acid protein (GFAP), a protein commonly associated with astrocytes, and that primary astrocyte cultures can contain GFAP-expressing cells that act as multipotent NSCs when transferred to neurogenic conditions. The relationship of GFAP-expressing NSCs to GFAP-expressing astrocytes is unclear, but has important implications. We compared the phenotype and neurogenic potential of GFAP-expressing cells derived from different CNS regions and maintained in vitro under different conditions. Multiple labeling immunohistochemistry revealed that both primary astrocyte cultures and adherent neurogenic cultures derived from postnatal or adult periventricular tissue contained subpopulations of GFAP-expressing cells that co-expressed nestin and LeX/CD15, two molecules associated with NSCs. In contrast, GFAP-expressing cells in similar cultures prepared from adult cerebral cortex did not express detectable levels of LeX/CD15, and exhibited no neurogenic potential. Fluorescence-activated cell sorting (FACS) of both primary astrocyte cultures and adherent neurogenic cultures for LeX/CD15 showed that GFAP-expressing cells competent to act as multipotent NSCs were concentrated in the LeX-positive fraction. Using neurosphere assays and a transgenic ablation strategy, we confirmed that the predominant NSCs in primary astrocyte and adherent neurogenic cultures were GFAP-expressing cells. These findings demonstrate that GFAP-expressing cells derived from postnatal and adult forebrain are heterogeneous in both molecular phenotype and neurogenic potential in vitro, and that this heterogeneity exists before exposure to neurogenic conditions. The findings provide evidence that GFAP-expressing NSCs are phenotypically and functionally distinct from non-neurogenic astrocytes.     

EGFP基因修饰大鼠胚胎中脑神经干细胞的建立

目的建立增强型绿色荧光蛋白（EGFP）基因修饰大鼠胚胎中脑神经干细胞。方法以质粒pEGFPNl转染培养第三代的大鼠胚胎中脑神经干细胞（mNSCs）,经G418筛选后,分别进行nestin免疫细胞化学鉴定和诱导分化后ρ-Ⅲ-／ubulin、GFAP、CNPase免疫细胞化学鉴定。结果EGFP在基因转染12h后开始表达,24h后明显增加,48h达到高峰,经G418筛选1月后有阳性克隆形成,EGFP基因修饰不影响大鼠胚胎mNSCs的增殖与分化。结论成功建立EGFP基因修饰大鼠胚胎mNSCs,为进一步开展帕金森病的细胞移植治疗研究奠定基础。     

Transplantation of neural stem cells for spinal cord injury]

Neural progenitor cells, including neural stem cells (NSCs), are an important potential graft material for cell therapeutics of damaged spinal cord. Here we used as a source of graft material a NSC-enriched population derived from human fetal spinal cord (Embryonic week 8-9) and expanded in vitro by neurosphere formation. NSCs labeled with BrdU (TP) or culture medium (CON) were transplanted into the adult marmoset spinal cord after contusion injury at C5 level. Grafted NSCs survived and migrated up to 7 mm far from the lesion epicenter. Double-staining with TuJ1 for neuron, GFAP for astrocyte, or CNPase for oligodendrocyte and BrdU revealed that grafted NSCs differentiated into neurons and oligodendrocytes 8 weeks after transplantation. More neurofilaments were observed in TP than those of CON. Furthermore, behavioral assessment of forelimb muscle strength using bar grip test and amount of spontaneous motor activity using infrared-rays monitoring revealed that the grafted NSCs significantly increased both of them compared to those of CON. These results indicate that in vitro expanded NSCs derived from human fetal spinal cord are useful sources for the therapeutics of spinal cord injury in primates.