大鼠骨髓基质干细胞诱导分化为肠神经细胞的体外研究

目的探讨不同方法诱导大鼠骨髓基质干细胞(BMSC)分化为肠神经细胞的可能性,并探索适宜的诱导方法。方法体外培养并鉴定BMSC,流式细胞法检测CD90和CD45。传代至第6代后行神经诱导分化。先以10ng/ml碱性成纤维生长因子(bFGF)预诱导24h,然后分2组：胶质细胞源神经营养因子(GDNF)组以10ng/ml GDNF,在胎肠培养基(FGCM)条件下诱导10d；维甲酸组以0．5 mmol/L维甲酸、10μmol/L ZnSO_4和FGCM诱导10d。免疫荧光法检测神经特异性烯醇化酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)、蛋白基因产物(PGP9．5)、一氧化氮合酶(nNOS)和血管活性肠肽(VIP)的表达。所得数据行t检验。结果流式细胞检测BMSC呈高表达,其标志为CD90,而不表达造血细胞标志CD45。诱导10d后,GDNF组和维甲酸组均有部分细胞在形态上表现出神经元样改变,免疫荧光染色NSE、NF、PGP9．5、nNOS、VIP阳性,GFAP阴性。GDNF组NF、PGP9．5、nNOS、VIP阳性比例显著高于维甲酸组(75．6％±8．4％比48．5±7．5％；57．7％±6．5％比35．7％±7．2％；46．6％±5．4％比30．5％±6．6％；72．3％±6．7％比40．4％±7．4％；P＜0．01)。结论在体外BMSC可被诱导分化为肠神经样细胞并表达肠神经递质,GDNF在胎肠培养基条件下诱导肠神经样细胞比例高于维甲酸。     

大鼠骨髓基质细胞体外培养转化为神经元细胞的实验研究

目的探讨体外原代培养骨髓基质细胞（BMSC）向神经元细胞转化情况.方法取成年Wistar大鼠BMSC,无血清和有血清培养基进行细胞克隆,获取骨髓间质干细胞（MSCs）,应用碱性成纤维细胞生长因子（bFGF）和表皮生长因子（EGF）进行细胞扩增及诱导分化.结果分离纯化后的BMSC经原代培养形成细胞克隆团,经传代后其数量明显增多,其分化细胞形态多样,包括神经元细胞、星形胶质细胞和少突胶质细胞,仍具有神经细胞所特有的性质.结论BMSC具有较强的自我更新能力和多分化潜能,在合适的环境条件下,可诱导分化出神经元细胞和神经胶质细胞,是较为理想的种子细胞.     

糖尿病早期视网膜靶组织顶盖前区神经退行性病变的观察

目的 动态观察糖尿病早期视网膜靶组织顶盖前区神经营养因子和神经胶质纤维酸性蛋白（GFAP）的表达，分析其靶组织神经元的改变和神经营养因子的变化与视网膜神经元变性的关系。方法 建立糖尿病模型后1周，2周，1月，分别用4％多聚甲醛心脏灌注大鼠，断头取顶盖，冰冻切片，免疫组化检测糖尿病早期视网膜靶组织顶盖前区脑原性神经营养因子（BDNF），神经生长因子（NGF）和GFAP的改变。实验中设正常对照（NC）组。结果 与NC组比较，糖尿病1周大鼠顶盖前区BDNF、NGF表达减少；糖尿病2周大鼠顶盖前区胶质细胞和细胞内NGFAP表达增加。结论 糖尿病早期视网膜靶组织顶盖前区神经细胞即已出现损害，神经营养因子表达减少，糖尿病早期视网膜靶组织顶盖前区神经退行性病变可能是糖尿病早期视网膜神经元变性的原因之一。     

脐血单个核细胞体外向神经干细胞的诱导及Foxg1基因的表达

目的探讨人脐血单个核细胞（UBC-MNCs）体外向神经干细胞（NSCs）的诱导分化机制及NSCs中Foxg1基因表达的意义。方法从脐血中分离出UBC—MNCs,用含hEGF、bFGF和B27因子的Neurobasa1培养基联合诱导其向NSCs方向分化,观察NSCs形态学及增殖分化特点;免疫组化法检测培养细胞中神经细胞标志抗原Nestin、NSE、GFAP的表达情况,BrdU标记靶细胞;RT-PCR法检测分离后、培养3、6、9、12d细胞中Foxg1基因的表达。结果UBC—MNCs可定向诱导分化为神经元样细胞,表达Nestin、NSE、GFAP标记抗原。Foxg1 mRNA在诱导前细胞中低表达,诱导后逐渐升高（P〈0．05）,6d达高峰,之后逐渐下降（P〈0．05）。结论体外定向诱导培养可获得UBC-MNCs源性NSCs,Foxg1基因是NSCs增殖分化关键调控因子,脐血可成为NSCs的新来源。     

神经调节蛋白在脑缺血中的保护作用

神经调节蛋白能调节胶质细胞的生长和分化以及小脑颗粒细胞沿胶质细胞的迁移,参与突触的形成,抑制脑缺血再灌注损伤诱导的神经元凋亡、胶质反应和炎症反应,诱导有关神经营养因子的表达,具有神经保护作用。     

大鼠胎血与骨髓间充质干细胞分化能力的体外研究

目的：比较大鼠胎血和骨髓中间充质干细胞（MSC）体外培养过程中的生长特性及体外诱导两者向神经元样细胞分化的异同。方法：采用标准Ficoll-hypague技术分离大鼠胎血骨髓的单个核细胞（MNC），收获MSC传代培养，流式细胞仪检测细胞的免疫表型。β-巯基乙醇、二甲基亚砜、叔丁基对羟基茴香醚诱导MSC向神经元分化，免疫细胞化学法检测其特异性标志巢蛋白（Nestin）、神经元特异性烯醇化酶（NSE）、胶原纤维酸性蛋白（GFAP）的表达。结果：2种MSC细胞形态、免疫表型无明显差异。定向诱导后，2种细胞均表达Nestin、NSE，但GFAP阴性。结论：大鼠胎血和骨髓MSC的细胞形态、生物学特性无明显差别；两者诱导分化为神经元碰细胞的能力无显著差异。胎血应是MSC的又一来源。     

胎脑神经干细胞的培养及鉴定

检测3个月胎龄人胎脑各个解剖部分神经巢蛋白（Nestin）的表达,分离神经干细胞进行培养,诱导其分化,利用免疫荧光细胞化学技术对培养的神经干细胞及分化细胞进行鉴定.结果 在人胎脑室管膜下区、海马、纹状体、丘脑、嗅球、皮层部位发现数量不等的Nestin阳性表达细胞,从海马和皮层组织分离培养得到的大量悬浮生长具有连续增殖能力的神经干细胞球,能够传代培养,表达神经干细胞的标志物Nestin,经诱导分化为神经元和胶质细胞.     

诱导型神经干细胞移植对颅脑创伤后神经营养因子分泌的影响

目的研究诱导型神经干细胞（iNSCs）移植对颅脑创伤（TBI）后神经营养因子分泌的影响。 方法采用自由落体脑打击装置制备雄性成年C57BL/6小鼠TBI模型，将神经功能缺损评分（NSS）4~8分者纳入TBI组，按照随机数字表法分为iNSCs移植组（12只）和磷酸盐缓冲液（PBS）处理组（9只）。同时设置假手术（sham）组（9只）。于TBI后12 h，用脑立体定向仪将含1×10⁶个iNSCs单细胞悬液或等体积PBS分别移植到TBI小鼠脑内，于移植后7 d处死动物。分别取脑组织mRNA（3只/组）行逆转录定量实时聚合酶链反应（RT-qPCR）和蛋白（6只/组）行酶联免疫吸附测定（ELISA），检测脑源性神经营养因子（BDNF）和胶质细胞源性神经营养因子（GDNF）表达量；取iNSCs移植组（3只）动物脑组织行免疫荧光染色，观察iNSCs移植物分泌BDNF和GDNF。 结果RT-qPCR示：相比sham组，TBI小鼠脑组织中Bdnf和Gdnf基因转录水平明显降低；相比PBS处理组，iNSCs移植组TBI小鼠脑组织中Bdnf和Gdnf基因转录水平明显升高，差异具有统计学意义（P<0.05）。ELISA示：相比sham组，TBI小鼠脑组织中BDNF和GDNF蛋白表达水平明显降低；相比PBS处理组，iNSCs移植组TBI小鼠脑组织中BDNF和GDNF蛋白表达水平明显升高，差异具有统计学意义（P<0.05）。免疫荧光染色示iNSCs移植组TBI小鼠脑内可见绿色荧光蛋白标记的iNSCs移植物迁移到达脑损伤区并表达BDNF和GDNF。 结论经脑立体定向移植iNSCs可在TBI后脑组织中合成分泌神经营养因子BDNF和GDNF，发挥神经营养作用。     

胶质细胞源性神经营养因子在胃肠道疾病中的研究进展

胶质细胞源性神经营养因子(GDNF)是来源于肠神经胶质细胞的一种重要功能性蛋白，在肠神经系统中发挥营养神经元、促进突触重塑、抗炎等重要作用。目前GDNF在胃肠道疾病进展中的作用逐渐引起重视。本文就GDNF及其配体、相关信号通路、与肠道内稳态的关系以及临床应用作一综述。     

神经营养因子对大鼠周围神经损伤后感觉神经元的保护作用观察

为研究神经营养因子对周围神经损伤后感觉神经元的保护作用，取Wistar大鼠20只，随机分成硅胶管内注射神经营养因子（NF组）和硅胶管内注入等量生理盐水（SAL组）两组，切断坐骨神经术后4周，用免疫组织化学法检测脊神经节内感觉神经元内一氧化氮合酶（iNOS）的表达，结果显示NF组同SAL组及正常对照神经元iNOS表达不同，NF组高于SAL组及正常对照，三组间有显性差异，说明神经营养因子对大鼠周围神经损伤后感觉神经元有保护作用。     

Pain-related differential expression of NGF, GDNF, IL-6, and their receptors in human vasculitic neuropathies

OBJECTIVE: Pain-related differential expressions of nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF) and interleukin-6 (IL-6), and their receptors were investigated in human vasculitic neuropathies. MATERIALS AND METHODS: The mRNA levels of pain-related neurotrophic factors, NGF, GDNF and IL-6, were examined in the sural nerves of 22 painful and non-painful patients with acute necrotizing vasculitic neuropathies, together with their concomitant soluble receptors (p75, GFR(alpha)-1 and IL-6R(alpha)). RESULTS: The mRNAs for these factors and receptors in the lesioned nerves were up-regulated to a variable extent in both groups. NGF mRNA expression was more closely correlated with that of p75 in painful neuropathy with relatively preserved large fiber density, compared with non-painful neuropathy, though the NGF mRNA level in painful neuropathy was lower than that in non-painful neuropathy. GDNF was closely associated with GFR(alpha)-1 in mRNA levels regardless of the pain state, but IL-6 was not associated with IL-6R(alpha). CONCLUSION: The differential expression of neurotrophic factors and their cognate soluble receptors in human vasculitic neuropathy suggests that NGF, which was effectively transferred to sensory axons with p75, may induce pain.     

The enteric glia and neurotrophic factors

Enteric glia cells (EGCs) play an important role in the maintenance of tissue integrity in the gastrointestinal tract. Thus, genetic ablation of glial fibrillary acidic protein (GFAP)-positive EGCs in mice induced fatal haemorrhagic jejuno-ileitis and led to death within a few days. The exact mechanisms of EGC to contribute to gut homeostasis remain enigmatic. Several lines of evidence implicate that the secretion of neurotrophic factors by EGC may be a part of the glial regulation of gut homoeostasis. The secretion of glia cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF) and transforming growth factor-beta (TGF-beta) contributes to the maintenance of epithelial integrity and the secretion of endothelins might be involved in vasoregulation. These new aspects of intestinal glial functions implicate new therapeutic strategies for diseases like Crohn's disease and irritable bowel syndrome.     

大鼠局灶性脑缺血再灌注后GDNF mRNA及其蛋白表达的变化

目的 观察大鼠脑缺血再灌注后缺血半暗带皮质脑源性神经营养因子(GDNF) mRNA和蛋白的表达变化.方法 采用线栓法制作局灶性脑缺血大鼠(MCAO)模型,用原位杂交法和免疫组化法观察脑缺血后3、6、12 h及l、3、7d共6个时间点GDNFmRNA及其蛋白的表达变化.结果 缺血半暗带GDNFmRNA及其蛋白的表达均于缺血灌注后3h开始明显上升,6h达高峰,12 h表达开始减弱(P＜0.05或P＜0.01),3-d后降至正常水平.结论 脑缺血再灌注后缺血半暗带GDNFmRNA及蛋白表达均明显增加可能参与了脑缺血后神经保护.     

大鼠局灶性脑缺血耐受胶质细胞源性神经营养因子的表达

目的探讨局灶脑缺血预处理对大鼠大脑中动脉栓塞(MCAO)后神经功能的影响及胶质细胞源性神经营养因子(GDNF)的表达。方法线栓法建立大鼠MCAO及预处理模型,Bederson神经功能评分观察大鼠神经功能,免疫组织化学和原位杂交技术检测GDNF表达。结果缺血预处理(IPC)可提高大鼠局灶性脑缺血后神经功能,且梗死周边区GDNF蛋白及mRNA表达水平高于对照组(P<0.05)。结论10 min IPC可产生缺血耐受,可能通过增加GDNF的表达而起脑保护作用。     

Neural differentiation of human mesenchymal stem cells: Evidence for expression of neural markers and eag K+ channel types

OBJECTIVE: Mesenchymal stem cells (MSCs) are multipotent cells that can self-renew, proliferate, and exhibit elevated cellular plasticity. To investigate their possible neural fate, we studied human mesenchymal stem cells (hMSCs) in different cell culture conditions from morphological, immunochemical, gene expression, and physiological points of view. MATERIALS AND METHODS: We tested hMSCs in three previously reported experimental conditions made of alpha-modified minimum essential medium (alpha-MEM)/1 mM beta-mercaptoethanol (betaME), 10 microM alpha-MEM/retinoic acid (RA) or alpha-MEM/2% dimethylsulfoxide (DMSO) + 200 microM beta-hydroxyanisole (BHA), respectively, and in a new experimental condition with neural progenitor maintenance medium (NPMM). RESULTS: hMSCs were isolated from bone marrow and expanded for several passages. In betaME, cells became immunoreactive for neuronal nuclear antigen (NeuN), neuron-specific enolase (NSE), Nestin, and glial fibrillary acidic protein (GFAP). In experimental conditions with RA and DMSO/BHA, hMSCs were NeuN and NSE-positive while in NPMM they were positive for GFAP and NSE. Untreated hMSCs showed a weak mRNA expression for microtubule-associated protein, NSE, and neurofilament protein-medium and GFAP, which strongly increased in NPMM-treated hMSCs. In the electrophysiological study, NPMM-differentiated hMSCs expressed two delayed rectifier K+ currents related to two ether-à-go-go K+ channels (eag1, eag2), which are fundamental for setting the negative resting potentials required for neuronal survival and basal cell activity. The two K+ channels were absent in undifferentiated hMSCs. These data were confirmed by real-time polymerase chain reaction. CONCLUSION: In our new culture condition, hMSCs acquired new morphological characteristics, neural markers, and electrophysiological properties, which are suggestive of neural differentiation. This might lead to clinical use of hMSCs in neural degenerative diseases.     

选择性深低温断血流对猴脑神经营养因子的影响

目的观察猴脑选择性深低温断血流复苏NGF、TrKAQA、GDNF的表达及其对神经营养因子表达的影响。方法 4～10岁健康恒河猴10只,动物随机分为三组:四血管(双侧颈总动脉及椎动脉)阻断冷灌注组(简称四血管组,n=5),两血管(双侧颈总动脉)阻断冷灌注组(简称两血管组,n=3),两血管(双侧颈总动脉)阻断等温灌注组(简称等温灌注组,n=3)。常温下临时阻断双侧颈部血管10 min后,通过一侧颈内动脉冷灌注(或等温灌注),同侧颈内静脉回流,阻断其他颈部血管,建立选择性脑局部体外循环通路,降低脑温至15.1℃±0.9℃,60 min恢复脑血流,实验动物自然复苏。等温灌注组及四血管组实验猴于灌注死亡后和两血管组实验猴饲养到术后12 w处死恒河猴,取脑、固定、脱水、石蜡包埋、切片,用NGF、TrkA、GDNF、Vimentin抗体进行免疫组织化学染色。对额叶恒定视野内NGF、TrkA、GDNF的阳性细胞进行记数并进行统计学分析。结果等温灌注组NGF、TrkA、GDNF有微量表达。四血管组NGF、TrkA、GDNF表达明显上调,与等温组和两血管组比较均有统计学意义(P<0.01)。两血管组NGF、TrkA、GDNF表达较明显上调,与等温灌注组比较有统计学意义(P<0.01)。结论 NGF、TrkA、GDNF表达上调,提示脑选择性超深低温断血流复苏过程中启动了脑自我保护机制,通过内源性神经营养因子表达水平上调,提高了神经细胞对缺血缺氧的耐受性,促进受损脑细胞的修复,是脑缺血的重要保护机制之一。     

Proinflammatory cytokines induce neurotrophic factor expression in enteric glia: a key to the regulation of epithelial apoptosis in Crohn's disease

OBJECTIVES: Imbalanced apoptosis of enterocytes is likely to be 1 of the mechanisms underlying Crohn's disease (CD). Apoptosis of enterocytes is regulated by glial-derived neurotrophic factor (GDNF), which is increased in CD. The cellular source of GDNF during gut inflammation is unclear. The aim of the study was to identify the source of GDNF in CD during gut inflammation. MATERIALS AND METHODS: Glial fibrillary acidic protein (GFAP), GDNF, and smooth muscle actin (SMA) was detected in the gut from patients with CD by immunohistochemistry. Cultured enteric glia cells (EGC) were labeled with anti-GFAP, anti-GDNF, and antibodies and a Golgi marker (anti-58K antibodies) after blocking Golgi export with monensin. Cultured EGCs were treated with interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, and lipopolysaccharides. Secretion of neurotrophic factors was detected by enzyme-linked immunosorbent assay. RESULTS: Mucosal GFAP-positive EGCs are increased in the colon of patients with CD. This type of glia but not subepithelial myofibroblasts expresses significant amounts of GDNF. In vitro GDNF is continuously secreted from cultured EGCs. The neurotrophic factor secretion could be stimulated by IL-1beta, tumor necrosis factor-alpha, and lipopolysaccharides in a time- and dose-dependent manner. The increased GDNF secretion by EGCs sustained for>12 hours after withdrawal of the proinflammatory cytokines. CONCLUSIONS: A mucosal GFAP expressing EGC population is dramatically increased in CD. This population is a major cellular source of the upregulated GDNF in the inflamed gut. Therefore, mucosal EGC may play a key role in protecting the gut epithelium and may contribute to reestablish the integrity of the injured epithelium.     

清热化瘀方对脑缺血再灌注大鼠GDNF mRNA及其蛋白表达的影响

目的观察大鼠脑缺血再灌注后脑内胶质细胞源性神经营养因子(GDNF)mRNA和蛋白的表达变化以及清热化瘀方对其的影响。方法采用线栓法制作局灶性脑缺血大鼠(MCAO)模型,用原位杂交法和免疫组化法观察脑缺血后3h、6h、12h及1d、3d、7d共6个时间点GDNF mRNA及其蛋白的表达变化。结果缺血半暗带GDNF mRNA及其蛋白的表达均于缺血灌注后3h开始明显上升,6h达高峰,12h表达开始减弱(P<0.05或P<0.01),3d后降至正常水平。清热化瘀方治疗后可以上调GDNF的表达。结论清热化瘀方可促进脑缺血后GDNF的表达,保护神经元。     

GDNF在慢传输型便秘大鼠胃壁中的表达以及外源性GDNF影响的研究

背景:慢传输型便秘(STC)主要以结肠传输功能障碍为特点,外源性胶质细胞系源性神经营养因子(GDNF)可改善其结肠动力。已有多项研究显示STC存在胃动力障碍,但胃壁GDNF异常表达以及外源性GDNF对胃动力障碍的作用目前尚无相关报道。目的:探讨GDNF在STC大鼠胃壁中的表达以及外源性GDNF的影响。方法:以大黄灌胃建立STC大鼠模型。将大鼠随机分为正常组、GDNF组、STC模型组和STC模型GDNF组,GDNF组和STC模型GDNF组大鼠尾静脉注射重组人GDNF,其余两组注射0.9%NaCl溶液。1周后处死大鼠,分别以RT-PCR和免疫组化法检测胃壁GDNF mRNA和蛋白表达。结果:STC模型GDNF组胃壁GDNF mRNA表达明显高于STC模型组(P0.05),STC模型组GDNF mRNA表达显著低于GDNF组(P0.05);STC模型GDNF组GDNF阳性表达与STC模型组相比差异无统计学意义(P0.05)。结论:长期使用大黄可减弱GDNF在STC大鼠胃壁组织中的表达;外源性GDNF可提高GDNF在STC大鼠胃壁组织中的表达。     

铁梭葡胺标记对骨髓干细胞向神经样细胞分化影响的实验研究

目的探讨铁梭葡胺(SHU555A)体外标记大鼠骨髓间充质干细胞(BMSCs)对其向神经样细胞分化的影响。方法在大鼠BMSCs培养液中分别加入10×稀释的SHU555A纳米颗粒10、20、40、80 μl,使铁的终浓度分别为14 μg/ml(14μg/ml标记组)、28 μg/ml(28 μg/ml标记组)、56 μg/ml(56 μg/ml标记组)和112 μg/ml(112 μg/ml标记组);另取无铁培养液的BMSCs作为对照组。各组BMSCs经DMSO+丁羟茴醚诱导6 h后,分别采用RT PCR和Western blot法检测神经上皮干细胞蛋白(nestin)、神经元特异性烯醇化酶(NSE)和神经胶质原纤维酸性蛋白(GFAP)的mRNA表达和nestin表达。结果与对照组比较,不同浓度标记组nestin、NSE和GFAP的mRNA均呈高表达,但差异无统计学意义(P0.05)。各组nestin表达差异无统计学意义(P0.05)。结论 14～112μg/ml的SHU555A标记浓度对大鼠BMSCs向神经样细胞分化无明显影响。