胰岛64KD蛋白胰酶消化片段抗原与胰岛素依赖型糖尿病

胰岛６４ＫＤ蛋白抗原（６４ＫＤａ）是胰岛素依赖型糖尿病（ＩＤＤＭ）的主要自身抗原，其抗体在ＩＤＤＭ患者中阳性率很高，６４ＫＤａ经胰酶消化后可产生５０ＫＤａ，４０ＫＤａ，３７ＫＤａ三片段，它们均与ＩＤＤＭ发病有紧密联系，尤其３７ＫＤａ对青少年发病型和快速进展型ＩＤＤＭ预测和诊断有重要价值。通过对三种片段与其它ＩＤＤＭ抗体的联系研究发现，５０ＫＤａ来源于谷氨酸脱羧酶，４０ＫＤａ的前体为胰岛细胞抗体（１     

机械法分离培养新生大鼠脊髓神经干细胞

目的 探讨新生大鼠脊髓源性神经干细胞的分离培养方法。方法 采用机械法及无血清培养技术从新生24h的SD大鼠脊髓中分离出脊髓源性神经干细胞,并采用免疫细胞化学方法鉴定神经干细胞和分化情况。结果 分离的细胞生长旺盛,单克隆化生成的细胞团,分离培养获得的细胞团呈Nestin强阳性,经胎牛血清诱导后可分化成为神经元、星形胶质细胞和少突胶质细胞。结论 利用机械法成功分离培养了新生大鼠脊髓神经干细胞,该细胞可连续传代,具备多向分化潜能。     

胰酶消化法培养大鼠胸主动脉平滑肌细胞

建立胰酶消化法培养大鼠胸主动脉平滑肌细胞,并和其他大鼠胸主动脉平滑肌细胞的培养方法相比较.在无菌条件下分离雄性Wistar大鼠胸主动脉血管中膜,剪碎中膜,在37℃用10mL0.25%胰酶消化大约3.5h.中止消化后,在100g条件下离心5min,收集细胞种入25cm2细胞培养瓶.结果发现,胸主动脉平滑肌细胞至少可以传20代以上,并且细胞形态、生长特点、平滑肌a-肌动蛋白表达不发生明显的改变.结果提示,与目前的平滑肌细胞培养方法相比,胰酶消化法培养平滑肌细胞的方法重复性好,原代培养的平滑肌细胞具有数量多、生长迅速的特点.     

胰岛64KD蛋白胰酶消化片段抗原与胰岛素依赖型糖尿病

胰岛６４ＫＤ蛋白抗原（６４ＫＤａ）是胰岛素依赖型糖尿病（ＩＤＤＭ）的主要自身抗原，其抗体在ＩＤＤＭ患者中阳性率很高，６４ＫＤａ经胰酶消化后可产生５０ＫＤａ，４０ＫＤａ，３７ＫＤａ三种片段，它们均与ＩＤＤＭ发病有紧密联系，尤其３７ＫＤａ对青少年发病型和快速进展型ＩＤＤＭ预测和诊断有重要价值。通过对三种片段与其它ＩＤＤＭ抗体的联系研究发现，５０ＫＤａ来源于谷氨酸脱羧酶，４０ＫＤａ的前体为胰岛细胞抗体（ＩＣＡ）５１２，３７ＫＤａ的前体为ｐｈｏｇｒｉｎ。     

脑梗死后神经干细胞的活化和移植

脑梗死后,缺血核心神经细胞的死亡不可避免。成年哺乳动物脑室下层(SVZ)和海马齿状回颗粒下层(SGZ)等保持着神经发生。脑梗死可促进神经发生。存在于这些部位的神经干细胞(NSC)具有自我更新和多向分化潜能。近年来,NSC的激活和向梗死灶周围迁移等机制逐渐为人们所认识,采取适当的措施激活脑内自然的神经发生、移植外源性NSC治疗脑梗死的研究,给脑梗死后缺损组织结构和功能的修复带来了希望。     

大鼠神经干细胞的分离培养和鉴定

目的建立神经干细胞分离、培养、鉴定技术.观察神经干细胞生长、增殖特点.方法利用无血清培养,从胚胎大鼠海马、纹状体等区分离神经干细胞,进行体外扩增培养、传代观察.采用荧光免疫细胞化学检测技术,观察鉴定神经干细胞.结果从胚胎大鼠海马、纹状体等区分离的细胞具有增殖能力,可进行传代培养,获得的细胞克隆中有nestin表达阳性细胞,显微镜下观察到典型的干细胞特征.结论用上述方法分离培养的神经干细胞具有自我更新和增殖能力,通过鉴定确为神经干细胞.     

莫沙必利联合胰激肽原酶治疗糖尿病神经源性膀胱疗效观察

目的探讨莫沙必利联合胰激肽原酶治疗糖尿病神经源性膀胱（DNB）的疗效及其安全性。方法将80例DNB患者随机分为观察组和对照组各40例,两组均采用基础治疗包括降糖、降压、营养神经、改善微循环及调节血脂等,观察组在基础治疗上加用莫沙必利和胰激肽原酶治疗,治疗21d后观察两组的血糖、血脂、血压和膀胱残余尿量改善情况。结果观察组治疗后总有效率明显高于对照组（P〈0．05）;两组治疗前后血糖、血压及血脂组间相比无明显差异（P〉0．05）,两组治疗后膀胱残余尿量明显少于治疗前（P〈0．05）,观察组治疗后膀胱残余尿量明显少于对照组（P〈0．05）;两组均未出现不良反应。结论莫沙必利联合胰激肽原酶治疗DNB能有效缓解患者的症状、体征,减少膀胱残余尿量,临床应用安全有效。     

不同种胰酶作用后形成的物质片段

胰腺分泌的各种胰酶能够进入小肠分解复杂的营养物质,在食物的消化吸收过程中起着至关重要的作用。不同种胰酶的含量及其作用的专一性处于精确的调控中,使得人类能够适应多种不同的饮食。慢性胰腺炎等多种胰腺疾病均可引起胰腺外分泌功能不全,从而导致消化不良、脂肪痢等症状。本文将针对胰酶对蛋白质、脂肪、碳水化合物等营养物质的消化作用以及不同种胰酶作用后形成的物质片段展开介绍,为多种胰腺疾病的早期诊断及治疗提供理论基础。     

人尿源干细胞的分离培养及体外成神经细胞分化研究

目的从正常成人尿液中分离提取间充质干细胞(MSC)来源的人尿源干细胞(hUSC),并研究比较其在不同诱导液中的体外成神经分化能力。方法提取正常成人来源hUSC,经体外培养、扩增,流式细胞术鉴定,用4种不同配方的成神经诱导液分别诱导hUSC向神经细胞分化,在诱导至第14天时,光镜下观察细胞形态改变,并用逆转录-聚合酶链反应(RT-PCR)方法鉴定Nestin、S100、β3-tublin和NF-200基因表达。采用SPSS 13.0统计软件对数据进行分析。组间比较采用t检验和方差分析。结果从正常成人提取的hUSC成功表达MSC表面标志物(CD27~+、CD44~+、CD73~+、CD90~+、CD31~-、CD34~-、CD45~-、HLA-DR~-)。hUSC在B、C、D成神经诱导液中诱导培养14 d后,细胞胞体回缩饱满、折光度变强,呈现神经元样突起。相比A成神经诱导液,B、C和D成神经诱导液中细胞Nestin、S100表达水平增高,B和D成神经诱导液中细胞β3-tublin、NF-200表达水平降低。其中C成神经诱导液中细胞Nestin表达水平高于B和D成神经诱导液中的细胞,B成神经诱导液中细胞S100表达水平高于C和D成神经诱导液中细胞,差异均具有统计学意义(P0.05)。结论 hUSC具备MSC特性;B、C、D成神经诱导液均有助于hUSC向神经类细胞方向分化,提示在优化诱导条件下,hUSC能向神经干细胞(NSC)及胶质细胞亚群分化。     

成年哺乳动物海马齿状回神经发生的研究进展

神经干细胞（NSCs）在一定条件下可增殖分化成神经元和神经胶质细胞,参与神经功能的修复过程,称之为神经发生。传统观念认为,神经发生主要存在于胚胎期。近年研究发现,成年哺乳动物的中枢神经系统也存在神经发生,生理状态下神经发生较少,而病理状态下神经发生较多。这为临床上神经损伤的修复提供了理论依据。现将成年哺乳动物海马齿状回神经发生的研究进展综述如下。     

Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice

Stem/progenitor cells from bone marrow and other sources have been shown to repair injured tissues by differentiating into tissue-specific phenotypes, by secreting chemokines, and, in part, by cell fusion. Here we prepared the stem/progenitor cells from human bone marrow (MSCs) and implanted athem into the dentate gyrus of the hippocampus of immunodeficient mice. The implanted human MSCs markedly increased the proliferation of endogenous neural stem cells that expressed the stem cell marker Sox2. Labeling of the mice with BrdUrd demonstrated that, 7 days after implantation of the human MSCs, BrdUrd-labeled endogenous cells migrated throughout the dorsal hippocampus (positive for doublecortin) and expressed markers for astrocytes and for neural or oligodendrocyte progenitors. Subpopulations of BrdUrd-labeled cells exhibited short cytoplasmic processes immunoreactive for nerve growth factor and VEGF. By 30 days after implantation, the newly generated cells expressed markers for more mature neurons and astrocytes. Also, subpopulations of BrdUrd-labeled cells exhibited elaborate processes immunoreactive for ciliary neurotrophic factor, neurotrophin-4/5, nerve growth factor, or VEGF. Therefore, implantation of human MSCs stimulated proliferation, migration, and differentiation of the endogenous neural stem cells that survived as differentiated neural cells. The results provide a paradigm to explain recent observations in which MSCs or related stem/progenitor cells were found to produce improvements in disease models even though a limited number of the cells engrafted.     

熟地含药血清对骨髓间质干细胞向神经细胞分化的影响

目的 观察熟地含药血清对骨髓间质干细胞(MSCs)向神经细胞分化的影响.方法 原代培养MSCs至第3代,并鉴定;熟地水煎剂分高、中、低3个剂量组对SD大鼠灌胃,10d后取大鼠血清加入培养至第三代的骨髓间质干细胞中诱导其分化,每组分别诱导24h,诱导后的细胞用免疫细胞化学法检测神经元特异性烯醇化酶(NSE)、巢蛋白(Nestin)、胶质纤维酸性蛋白(GFAP)三种蛋白.结果 原代培养鉴定为MSCs;熟地含药血清能诱导MSCs向神经细胞分化,诱导后与对照组比较,高、中、低3个剂量组细胞NSE,Nestin的表达率都较高(P＜0.05),且高、低剂量组之间比较有统计学意义(P＜0.05);但GFAP表达极低.表明所分化的细胞为神经细胞.流式细胞仪检测结果 显示,β-巯基乙醇对细胞诱导分化能力强而促细胞增殖能力弱.结论 经典的培养方法,可分离出相对较多、纯度较高、生长状态良好的MSCs细胞;熟地含药血清能够促进MSCs向神经细胞的分化.     

Betacellulin promotes cell proliferation in the neural stem cell niche and stimulates neurogenesis

Neural stem cells (NSCs) reside in specialized niches in the adult mammalian brain, including the subventricular zone and the dentate gyrus, which act to control NSC behavior. Among other cell types within these niches, NSCs are found in close proximity to blood vessels. We carried out an analysis of the interaction between endothelial cells and NSCs, and show that betacellulin (BTC), a member of the EGF family and one of several signaling molecules made by the former, induces NSC proliferation and prevents spontaneous differentiation in culture. When infused into the lateral ventricle, BTC induces expansion of NSCs and neuroblasts, and promotes neurogenesis in the olfactory bulb and dentate gyrus, whereas specific blocking antibodies reduce the number of stem/progenitor cells. BTC-null mice are less able to regenerate neuroblast numbers compared with WT littermates following depletion of proliferating cells using cytosine-β-d-arabinofuranoside. BTC acts via both the EGF receptor, located on NSCs, and ErbB4, located on neuroblasts, with the latter explaining why its effects are distinct from those of EGF itself. Our results suggest that BTC could be a good candidate to aid regenerative therapies.     

Identifying and tracking neural stem cells

Hematopoietic stem cells, unlike neural stem cells, can be readily identified and isolated from developing and adult cell populations using positive and negative selection criteria. Isolating stem cells and progenitor cells from neural tissue has been more difficult because of difficulties in separating cells in solid tissue, the limited numbers of stem cells that persist in the adult, and the paucity of rigorously characterized markers. Nevertheless, strategies that have worked successfully in hematopoietic stem cell isolation can be adapted to isolate multiple classes of stem and progenitor cells from neural tissue. Neural stem cells also share cellular and molecular properties with other stem cell populations that may serve as surrogate identifiers of multipotentiality. Such potential markers are described. Unlike hematopoietic stem cells, tracking neural cells after transplantation is both necessary and more difficult. It will therefore be necessary to develop invasive and non-invasive strategies to follow transplanted cells and develop useful quantifiable readouts. Some potential strategies are described and current results are discussed.     

β-淀粉样蛋白对培养胎鼠大脑神经干细胞作用的研究

目的探讨β淀粉样蛋白(amyloidbetaprotein,Aβ)对神经干细胞的影响。方法实验分为对照组和Aβ140组(Aβ140浓度为0.01、0.05、0.1、1、5、10、20μmolL),分别培养60h,测定神经干细胞培养上清中一氧化氮(NO)和乳酸脱氢酶(LDH)含量,免疫组织化学方法观察细胞培养物Bcl2、Bax表达情况;培养60h后,改变培养条件,诱导神经干细胞分化72h,免疫组织化学染色观察培养物微管相关蛋白(MAP2)、胶质纤维酸性蛋白(GFAP)阳性细胞量的变化。结果Aβ140作用神经干细胞60h后,培养液中NO含量增高,LDH活性升高,与对照组比较差异有显著性意义。免疫组织化学染色结果,随着Aβ140浓度不断增高,Bcl2含量降低,与对照组比较有显著性差异,Bax含量增高,与对照组比较有显著性差异;神经干细胞分化后,MAP2、GFAP阳性细胞量均降低,与对照组比较有显著性差异。结论Aβ可导致神经干细胞的细胞毒性增加,细胞膜完整性降低,Bcl2表达下调,Bax表达上调。     

A method for enzymatic dissociation of cells from isolated pancreatic islets

Summary An enzymatic method for isolation of single cells from the islets of Langerhans is described. The isolated cells appeared well preserved and survived for at least 7 days when maintained in culture. The dry mass of the isolated islet cells was found to be decreased 30 min after administration of alloxan to obese-hyperglycemic mice. Isolated individual islet cells from obese-hyperglycemic mice had a higher dry mass than those from their lean litter mates. Traduzione a cura di G. U.     

Polyclonal fluctuation of lentiviral vector-transduced and expanded murine hematopoietic stem cells

Gene therapy has proven its potential to cure diseases of the hematopoietic system. However, severe adverse events observed in clinical trials have demanded improved gene-transfer conditions. Whereas progress has been made to reduce the genotoxicity of integrating gene vectors, the role of pretransplantation cultivation is less well investigated. We observed that the STIF (stem cell factor [SCF], thrombopoietin [TPO], insulin-like growth factor-2 [IGF-2], and fibroblast growth factor-1 [FGF-1]) cytokine cocktail developed to effectively expand murine hematopoietic stem cells (HSCs) also supports the expansion of leukemia-initiating insertional mutants caused by gammaretroviral gene transfer. We compared 4 protocols to examine the impact of prestimulation and posttransduction culture in STIF in the context of lentiviral gene transfer. Observing 56 transplanted mice for up to 9.5 months, we found consistent engraftment and gene-marking rates after prolonged ex vivo expansion. Although a lentiviral vector with a validated insertional-mutagenic potential was used, longitudinal analysis identifying > 7000 integration sites revealed polyclonal fluctuations, especially in "expanded" groups, with de novo detection of clones even at late time points. Posttransduction expansion in STIF did not enrich clones with insertions in proto-oncogenes but rather increased clonal diversity. Our data indicate that lentiviral transduction in optimized media mediates intact polyclonal hematopoiesis without selection for growth-promoting hits by posttransduction expansion.