成体神经干细胞培养方法的建立

目的 探讨体外建立培养成体神经干细胞(MSC)的方法.方法 从6周龄大鼠脑组织中分离神经干细胞,用神经干细胞培养液[DMEM/F12培养液添加1%N2、2%B27、20 μg/L表皮生长因子(EGF)和20μg/L碱性成纤维细胞生长因子(bFGF)]使其稳定增殖,10%胎牛血清诱导其贴壁分化.倒置显微镜下观察神经干细胞形态学变化;流式细胞仪检测神经干细胞表面标记物巢蛋白(Nestin)、神经元特异性烯醇酶(NSE,成熟神经元的特异性标志)、半乳糖脑苷脂(Galc-C,成熟少突胶质细胞的标记物)的表达.结果 分离所得细胞能在体外传代培养,流式细胞仪检测显示Nestin阳性细胞为97.6%,细胞经胎牛血清诱导分化后能形成NSE、Galc-C阳性细胞.结论 采用无血清的神经干细胞培养液能培养出具有分化潜能的成体神经干细胞.     

绿色荧光蛋白转基因神经干细胞脑出血大鼠脑内移植后血管生成与神经形态学的改变

目的 观察绿色荧光蛋白转基因神经干细胞(NSCs/GFP)脑出血大鼠脑内移植后血管生成与神经形态学改变.方法 分离、培养及纯化NSCs/GFP,并进行体外诱导分化.注射动脉血制作大鼠脑出血模型,3d后将NSCs/GFP注入血肿同侧尾状核,观察NSCs/GFP在脑内分化情况以及大鼠不同时间点行为学的变化.结果 成功分离、培养及鉴定NSCs/GFP,体外诱导分化为内皮细胞和平滑肌细胞；移植入脑出血大鼠脑内后,能促进其肢体功能恢复(P＜0.05),且能分化为神经元、星型胶质细胞、内皮细胞及平滑肌细胞.结论 NSCs/GFP移植入脑出血大鼠脑内能改善其运动功能,并有生成血管与神经的能力.     

胚胎大鼠脊髓神经干细胞体外培养和分化的研究

目的探讨脊髓源性神经干细胞的培养方法和体外分化特性. 方法从孕龄15天的胚胎大鼠脊髓组织中分离获得神经干细胞,采用含表皮生长因子及碱性成纤维细胞生长因子的无血清限定性培养基培养并进行干细胞体外分化,细胞免疫荧光化学方法鉴定分化结果. 结果胚胎脊髓中可分离得到大量的神经干细胞,培养10天左右可获得干细胞球;用细胞免疫荧光化学法鉴定干细胞分化,可见其中含有神经元和星形胶质细胞. 结论无血清限定性培养基是脊髓源性神经干细胞的良好培养基;脊髓源性神经干细胞体外可诱导分化为神经元和神经胶质细胞.     

大鼠雪旺细胞对脊髓源性神经干细胞生长及分化的影响

目的 观察雪旺细胞(SCs)在体外对脊髓来源的神经干细胞(NSCs)生长及分化的影响.方法 分别从胎鼠脊髓和出生24 h内的SD大鼠坐骨神经分离培养NSCs及SCs并鉴定.实验分成对照组:NSCs单独培养组;A组:脑源性神经营养因子(黼)与NSCs共培养组;B组:SCs与NSCs共培养组;C组:BDNF+SCs与NSC共培养组.观察NSCs的形态学变化,计数并测量细胞团的数量及直径.利用免疫细胞化学方法检测神经元样细胞的表达并计算其百分率及阳性细胞突起的平均长度.结果 来源于新生大鼠脊髓组织的NSCs能够表达NSCs的标志物神经巢蛋白(Nestin),NSCs可分化为神经元及星形胶质细胞;72 h,A、B、C组细胞团的数量及细胞团的平均直径,均大于对照组(P＜0.05);传代培养3 d、7 d、14 d的NAP-2染色阳性神经元样细胞所占的百分率及阳性细胞突起平均长度均大于对照组(P＜0.05).结论 大鼠SCs在体外能够促进脊髓来源的NSCs生长并诱导其定向分化.     

组织-细胞共培养诱导神经干细胞定向分化的研究

目的 探讨大鼠上丘组织-神经干细胞(NSCs)共培养对细胞分化的影响.方法 采用无血清选择培养的方法获得上丘源NSCs,单细胞克隆传代培养并进行形态学观察.使用组织-细胞共培养系统将不同年龄大鼠上丘组织与NSCs共培养,观察NSCs分化过程.对分化细胞进行鉴定,并与NSCs的自然分化过程进行比较.结果 从胚鼠上丘中获得的细胞呈球形悬浮生长,可形成单细胞克隆,具有多向分化能力,经免疫荧光鉴定证实为NSCs.新生大鼠上丘组织与NSCs共培养可以促进NSCs的分化,并诱导分化出Thy1.1阳性的视网膜神经节细胞,阳性细胞比率为19.97%(273/1094).而神NSCs的自然分化或与成年大鼠上丘组织共培养后均未发现Thy1.1阳性细胞.结论 从胚胎大鼠上丘可以分离培养出NSCs,与新生大鼠的上丘组织共培养可以诱导NSCs分化为视网膜神经节细胞.     

诱导成鼠骨髓源神经干细胞的实验研究

目的：探讨体外诱导成鼠骨髓源神经干细胞的可行性。方法：以含20ng／ml碱性成纤维生长因子(basic fibroblast growth factor，bFGF)与表皮生长因子(epidermal growth factor，EGF)的无血清培养液培养成鼠骨髓细胞，待细胞球形成后进行传代培养与单细胞克隆试验．以含100ng／ml bFGF培养液诱导分化，细胞免疫组化染色进行细胞鉴定。结果：骨髓细胞在诱导条件下形成表达Nestin抗原的细胞球．亦表达造血干细胞标志性抗原CD90。分离成单细胞后很快形成新的细胞球，并可分化出神经元与胶质细胞。结论：骨髓细胞可在体外诱导为能自我增殖的神经干细胞。     

神经干细胞移植对大鼠脊髓损伤后BDNF与GAP-43基因表达的影响

目的:研究海马源性神经干细胞(NSCs)移植对大鼠脊髓损伤(SCI)后生长相关蛋白43(GAP-43)及脑源性神经营养因子(BDNF)基因表达的影响,探讨神经干细胞移植修复大鼠脊髓损伤的机制.方法:NSCs提取自新生胎鼠的海马区,经过培养及鉴定.实验分为3组:NSCs移植组、DMEM填充组、正常对照组.大鼠SCI后第7d移植NSCs,应用RTPCR法观察NSCs移植后,大鼠脊髓损伤区GAP-43和BDNF基因表达的变化.结果:NSCs移植组较单纯损伤组明显增强了GAP-43mRNA与BDNFmRNA的表达.结论:NSCs移植后改变脊髓损伤区的微环境,上调BDNFmRNA,促进GAP-43mRNA的表达,是修复脊髓损伤的机制之一.     

神经干细胞移植治疗糖尿病大鼠神经源性膀胱的实验研究

目的 研究神经干细胞(Neural Stem Cells,NSCs)移植对于治疗糖尿病引起的神经源性膀胱(diabetic neurogenic bladder,DNB)的实验疗效.方法 SD大鼠腹腔注射链脲佐菌素(Streptozotocin,STZ)制造出糖尿病大鼠神经源性膀胱模型,用胰岛素控制血糖后,将从新生SD大鼠海马组织分离培养出的NSCs,通过股静脉注射、膀胱肌肉注射移植入2组糖尿病SD大鼠神经源性膀胱模型中,通过尿流动力学观察移植后8周各组SD大鼠的排尿情况.结果 膀胱肌肉注射NSCs,能更好修复糖尿病大鼠神经源性膀胱的受损神经,改善排尿功能.结论 NSCs移植可作为糖尿病大鼠DNB的一种治疗方法.     

神经干细胞移植对大鼠脊髓损伤后BDNF与GAP-43基因表达的影响

目的：研究海马源性神经干细胞(NSCs)移植对大鼠脊髓损伤(SCI)后生长相关蛋白43(GAP-43)及脑源性神经营养因子(BDNF)基因表达的影响，探讨神经干细胞移植修复大鼠脊髓损伤的机制。方法：NSCs提取自新生胎鼠的海马区，经过培养及鉴定。实验分为3组：NSCs移植组、DMEM填充组、正常对照组。大鼠SCI后第7d移植NSCs，应用RT-PCR法观察NSCs移植后，大鼠脊髓损伤区GAP-43和BDNF基因表达的变化。结果：NSCs移植组较单纯损伤组明显增强了GAP-43mRNA与BDNFmRNA的表达。结论：NSCs移植后改变脊髓损伤区的微环境，上调BDNFmRNA，促进GAP-43mRNA的表达，是修复脊髓损伤的机制之一。     

褪黑素对大鼠脊髓神经干细胞增殖和分化的影响

目的:研究不同浓度的褪黑素(melatonin,MT)对胚胎大鼠脊髓神经干细胞(neuralstemcells,NSCs)增殖和分化的影响。方法:取孕14d胚胎大鼠脊髓,分离并原代培养脊髓NSCs。传四代后分为对照组和实验组,实验组分别加入不同浓度的MT(100μmol/L、1μmol/L及10nmol/L)。采用细胞计数结合免疫荧光检测法,观察脊髓NSCs的生长增殖及分化状况,并作统计学分析。结果:100μmol/L和1μmol/L的浓度的MT均能促进脊髓NSCs增殖;10nmol/L时则抑制脊髓NSCs的增殖;3种浓度的MT均能使脊髓NSCs向神经元分化的比率增加,对照组和实验组的神经元分化比率分别为(23.6±2)%、(45.3±1)%、(42.5±2)%、(50.7±2)%。结论:3种不同浓度的褪黑素对大鼠脊髓NSCs体外增殖有着不同的作用,但都有促进其向神经元分化的作用。     

成纤维细胞生长因子和表皮生长因子及复合因子对兔ACL、MCL体外增殖作用

目的观察酸性成纤维细胞生长因子(acid fibroblast growth factor，aFGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)和表皮生长因子(epidermal growth factor，EGF)以及复合因子对兔前交叉韧带(anterior cruciate ligament，ACL)和内侧副韧带(medial collateral ligament，MCL)的促增殖作用。方法分离传代培养10周龄新西兰大白兔骨关节韧带的ACL和MCL的成纤维细胞，接种96孔板，并加入浓度为0(对照组)、1、5、10、50、100ng／ml的aFGF或bFGF，浓度为0(对照组)、1．56、3．13、6．25、12．50、25、50ng／ml的EGF，单独或aFGF EGF两种因子联用与细胞(n=4)共同培养48h，以XTT方法测定其促细胞增殖作用。结果3种生长因子单独应用对ACL和MCL都有促进作用，aFGF对两种细胞均存在量效关系；bFGF 1ng／ml，EGF 5ng／ml对ACL作用最好，而对MCL则是bFGF和EGF均存在量效关系。浓度为5ng／ml的aFGF与50ng／ml的EGF联合1ng／ml aFGF与3．13ng／mlEGF作用于ACL或MCL均有协同作用。结论3种生长因子对ACL和MCL均有促进作用，单独应用aFGF或联用EGF优于单一因子促进兔ACL和MCL细胞的增殖，并且提示低浓度的aFGF联用EGF优于单一生长因子。     

全反式维甲酸对鼠胚神经干细胞增殖和分化的作用

目的 观察不同浓度的全反式维甲酸(all-trans-retinoic acid,ATRA)在鼠胚神经干细胞(neural stemcells,NSCs)增殖和分化中的作用,寻找促NSCs分化为神经元的较佳ATRA浓度. 方法 分离孕12～16 d(平均15 d)胚胎大鼠脑皮层,用无血清但含神经生长因子(20 ng/mL bFGF、20 ng/mL EGF)的培养液悬浮培养,细胞接种密度为1×106/mL,7 d传代.取第3代NSCs,分别在含0.5、1.0、5.0和10.0 μmol/L ATRA(实验组,分别为A、B、C、D组)及不含ATRA(对照组,E组)的DMEM/F12完全培养基中培养,于培养第1、2、3、4、5、6及7天倒置相差显微镜下计数形成的细胞球数;于培养后1、3、5、7及9 d采用BrdU免疫荧光染色,分析各组细胞增殖效应.另取第3代NSCs,5?S培养基调整细胞密度为1×106/mL,接种至6孔板内,分别加入0.5、1.0、5.0和10.0 μmol/L ATRA,作为各实验组(即A、B、C、D组),对照组不含ATRA(E组),于培养后3、5及7 d采用免疫荧光双标染色和流式细胞仪检测各组细胞分化为神经元的比例. 结果 培养后1、2、3、4、5、6及7 d,各实验组增殖细胞球数目接近(P＞0.05),但均明显少于对照组,且差异有统计学意义(P＜0.05);各实验组在培养后1、3、5、7、9dBrdU阳性细胞球增多,组间差异无统计学意义(P＞0.05);对照组各时间点BrdU阳性细胞球明显多于实验组,差异有统计学意义(P＜0.05).各实验组ATRA促使NSCs分化为神经元的比例明显增高,NSE阳性细胞分化率各组间差异有统计学意义(P＜0.05),其中B组最高,在培养3、5、7 d分别为29.46%±0.47%、47.25%±0.46%、66.81%±0.57%,而E组NSCs主要分化为星形胶质细胞,NSE阳性细胞分化率在培养3、5、7 d分别为11.11%±0.59%、14.10%±0.32%、15.92%±0.70%.流式细胞仪检测显示,培养后3、7 d,促神经元分化比例各实验组与对照组比较差异均有统计学意义(P＜0.05). 结论 ATRA具有显著的促NSCs分化为神经元的作用,1.0 μmol/L ATRA诱导分化效果最佳.     

Morphological differentiation of astroglial progenitor cells from EGF-responsive neurospheres in response to fetal calf serum,basic fibroblast growth factor,and retinol

Procurement of multipotential neuroglial stem cells is possible with the addition of epidermal growth factor (EGF). Stem cells will differentiate into neurons and glia upon the removal of EGF from the culture medium. We have previously characterized the neuronal differentiation of stem cells derived from long-term cultured nonpassage neurospheres. In the current study, we (1) characterize the morphological differentiation of the astroglial progenitor cell from 3-mo-old neurospheres, (2) examine whether the astroglial progenitor cells from neurospheres of different brain areas exhibit different differentiation responses to the same exogenous signals, and (3) test the effects of basic fibroblast growth factor (bFGF) and retinol on differentiation. Cerebral cortex, striatum, and mesencephalon cells were obtained from Embryonic Day 14 (E-14) rat fetuses and were dissociated for the procurement of neurospheres in chemically defined medium supplemented with EGF. After 3 mo in culture, the neurospheres, derived from each of the three brain areas, were subcultured into three groups on chamber slides: (1) basal medium, (2) the basal medium plus 20 ng/mL bFGF, and (3) the basal medium plus 10 μM retinol. Phenotypic expression of astroglial cells was examined after 14 days subculture. Our findings indicate that the 3-mo-old cultured nonpassage neurospheres contained numerous multipotential stem cells that stained positive with nestin, and that environmental factors played an important role in influencing the differentiation of astroglial progenitor cells. As detected by glial fibrillary acid protein (GFAP), astroglial progenitor cells turned into protoplasmic astrocytes in the FCS-containing basal medium, fibrous astrocytes in the presence of bFGF, and spindle-shaped astrocytes in the presence of retinol. There were no noticeable differences in differentiation among astroglial progenitor cells of the various brain region-derived neurospheres in any of the three medium conditions. Peculiar varicosity and growth cone-like structures on the long slender GFAP-positive processes suggest that neuroblasts and glioblast may share common morphologies, features, or common progenitor cells during initial differentiation in vitro.     

碱性成纤维细胞生长因子和表皮生长因子对骨骼肌源性干细胞的促增殖效应

目的观察碱性成纤维细胞生长因子（basic fibroblast growth factor，bFGF）和表皮生长因子（epidermal growth factor，EGF）单独及联合应用对骨骼肌源性干细胞（muscle derived stem cells，MDSCs）生长的影响。方法取出生24h内的昆明小鼠15只，采用连续预贴壁法从小鼠后肢肌分离培养MDSCs，用含2％胎牛血清的DMEM培养基促进其向骨骼肌细胞分化。取原代MDSCs及MDSCs分化细胞，采用免疫细胞化学染色检测干细胞标志Sca-1和骨骼肌细胞标志α-Sarcomeric肌动蛋白的表达。HE染色观察细胞肌管形成。MTT比色法检测不同浓度（6．25、12．50、25．00、50．00、100．00ng／ml）的bFGF和EGF单独应用96h对MDSCs增殖的影响以及二者（100．00ng／ml）联合作用24、48、72和96h对MDSCs增殖的影响。结果从新生小鼠后肢肌成功分离培养MDSCs，免疫细胞化学染色90％以上的MDSCs呈Sca-1阳性，分化形成的肌管呈α—Sarcomeric肌动蛋白阳性。HE染色可见肌管形成。bFGF、EGF对MDSCs的促增殖效应随浓度的增加而增加。与阴性对照组比较，bFGF于12．50ng／ml出现促增殖效应（P〈0．05）；25．00ng／ml组与12．50ng／ml组比较，作用提高（P〈0．01）；50．00、100．00ng／ml组较25．00ng／ml组无明显提升（P〉0．05）；EGF的作用与bFGF类似，但于50．00ng／ml时趋于饱和。与阴性对照组比较，EGF于72h、bFGF于96h表现促增殖效应（P〈0．01），而二者联合应用于24h即表现促增殖效应（P〈0．01），并于48、72和96h增殖效应均较单独应用显著提高（P〈0．05）。结论bFGF和EGF都能促进MDSCs的增殖，联合作用更快、更强。     

碱性成纤维细胞生长因子对成骨细胞粘附特性影响的实验研究

目的 针对骨组织工程中成骨细胞与基质材料间促粘附问题,研究碱性成纤维细胞生长因子（ｂＦＧＦ）对成骨细胞粘附特性的影响。方法 取兔骨髓基质干细胞来源的成骨细胞体外培养,分别用５、１０、５０、１００及２００ｎｇ／ｍｌ浓度的ｂＦＧＦ诱导培养２４小时作为实验组;不含ｂＦＧＦ的培养基作为对照组。观察接种后０．５、１、２、４、及８小时各时间点成骨细胞粘附情况,体现学计量粘附细胞数量。结果 １０ｎｇ／ｍｌｂＦＧ     

骨形成蛋白-2诱导脊髓神经干细胞分化的实验研究

目的：研究不同浓度的骨形成蛋白-2(bone norphogenetic protein-2，BMP-2)对脊髓源性神经干细胞(neural stem cells，NSCs)诱导分化成为神经细胞(神经元和神经胶质细胞)的影响。方法：取孕14d的胚胎小鼠脊髓，原代培养出脊髓NSCs。培养四代后，分别在碱性成纤维细胞生长因子(bFGF)存在与否的不同条件下，加入不同浓度的BMP-2．观察细胞的增殖、分化情况，用免疫组织化学的方法进行鉴定，并进行统计学分析：结果：低浓度的BMP-2(1～10ng／ml)与bFGF(20ng／ml)联合作用，可促进脊髓NSCs向神经元和星形胶质细胞分化，抑制其向少突胶质细胞分化。较高浓度BMP-2(100ng／ml)可抑制脊髓NSCs的增殖甚至导致细胞死亡。结论：脊髓NSCs在低浓度BMP和bFGF联合作用下能有效地分化成具有多种特性的神经样细胞，并与脊髓具有良好的同源性，可为脊髓损伤的修复研究提供良好的细胞学基础。     

Differentiation of adult human bone marrow mesenchymal stem cells into Schwann-like cells in vitro

Bonemarrowmesenchymalstemcells, alsocalledbonemarrowstromalcells (BMSCs), areisolatedfrombonemarrow, andtheycanmultiplyinvitroanddifferentiateintoosteogeniccells,chondrocytes, adipocytes, musclecellsandneuralcells.1 5 RecentstudieshavedemonstratedthatBMSCsfromadultratscoulddifferentiateintoSchwann likecellsinspecificconditions.6, 7 BMSCsmayhavepotentialapplicationforautologousneuraltransplantation. Inthisstudy, wetrytoinvestigatethedifferentiativecapabilityofadulthumanBMSCsintoSchwann l…     

In vitro screening of exogenous factors for human neural stem/progenitor cell proliferation using measurement of total ATP content in viable cells

One of the newest and most promising methods for treating intractable neuronal diseases and injures is the transplantation of ex vivo-expanded human neural stem/progenitor cells (NSPCs). Human NSPCs are selectively expanded as free-floating neurospheres in serum-free culture medium containing fibroblast growth factor 2 (FGF2) and/or epidermal growth factor (EGF); however, the culture conditions still need to be optimized for performance and cost before the method is used clinically. Here, to improve the NSPC culture method for clinical use, we used an ATP assay to screen the effects of various reagents on human NSPC proliferation. Human NSPCs responded to EGF, FGF2, and leukemia inhibitory factor (LIF) in a dose-dependent manner, and the minimum concentrations eliciting maximum effects were 10 ng/ml EGF, 10 ng/ ml FGF2, and 5 ng/ml LIF. EGF and LIF were stable in culture medium without NSPCs, although FGF2 was degraded. In the presence of human NSPCs, however, FGF2 and LIF were both degraded very rapidly, to below the estimated minimum concentration on day 3, but EGF remained above the minimum concentration for 5 days. Adding supplemental doses of each growth factor during the incubation promoted human NSPC proliferation. Among other supplements, insulin and transferrin promoted human NSPC growth, but progesterone, putrescine, selenite, D-glucose, and lactate were not effective and were cytotoxic at higher concentrations. Supplementing with conditioned medium from human NSPCs significantly increased human NSPC proliferation, but using a high percentage of the medium had a negative effect. These findings suggest that human NSPC culture is regulated by a balance in the culture medium between decreasing growth factor levels and increasing positive or negative factors derived from the NSPCs. Thus, in designing culture conditions for human NSPCs, it is useful to take the individual properties of each factor into consideration.     

Growth inhibition of epidermal growth factor-stimulated human glioblastoma cells by nicardipine in vitro.

BACKGROUND: The objective of this work was to observe and analyze the effects of epidermal growth factor (EGF) and the calcium channel antagonist nicardipine on the growth of U251MG, a human malignant glioma cell line, which have high-affinity EGF receptors. METHODS: The growth effects of EGF and nicardipine on U251MG cultured in serum-free and serum-supplemented (10% fetal bovine serum, FBS) medium respectively were observed by MTT colorimeritric analysis. RESULTS: EGF significantly enhanced the growth of U251MG cells in a dose-dependent manner in serum-free medium. The maximal effect was seen at 20 ng/ml. The effects of EGF approximated those obtained in 10% FBS. Nicardipine decreased U251MG cell proliferation, especially in serum-supplemented medium, and completely blocked the growth-stimulated effects of EGF. The combined effects of EGF (10 ng/ml) and nicardipine equaled those of nicardipine alone. CONCLUSIONS: When serum was absent, the U251MG cells showed a pronounced mitogenic response to EGF in a dose-dependent manner, which approximated that achieved with 10% FBS. The addition of serum obscured this effect. Nicardipine suppressed the growth of U251MG cells and completely blocked the growth-stimulated effects of EGF may suggest a possible role of this drug as adjuvant therapy for human malignant gliomas.     

Autocrine regulation of DU145 human prostate cancer cell growth by epidermal growth factor-related polypeptides

The DU145 human prostate cancer cell line possesses epidermal growth factor (EGF) receptors and synthesizes both EGF and the related polypeptide transforming growth factor-alpha (TGF-alpha). A monoclonal antibody to the EGF receptor was used to determine whether these characteristics were indicative of a functional autocrine regulatory system. This antibody competed effectively with [125I]EGF for binding to DU145 cell binding sites over a 1 x 10(-11) to 1 x 10(-7) M concentration range, and did so with a capability similar to that of the two natural ligands. It inhibited growth of these cells in both 3% fetal bovine serum-supplemented and serum-free medium; in experiments with incubation times of 3-5 days there was a 45-50% reduction in cell number. Growth suppression by the EGF receptor blockade of cells plated at a density of 1.5 x 10(4) cells/ml/well was reversed competitively by the addition of EGF to the medium; 0.3 nM completely eliminated the inhibitory effect of a 1 x 10(-9) M antibody concentration. It is concluded that DU145 cell growth is regulated by an EGF-mediated autocrine loop.