时间因素对神经干细胞向γ-氨基丁酸能神经元分化的影响

目的探讨时间因素对体外培养的神经干细胞向γ氨基丁酸(gammaaminobutyricacid,GABA)能神经元分化的影响。方法体外培养神经干细胞并加以鉴定。利用反义寡核苷酸诱导神经干细胞向GABA能神经元分化,通过免疫荧光染色计算不同时间点的GAD65阳性细胞率。结果体外培养获得神经干细胞,染色可见神经球及分化细胞中分别有nestin、NF200、GFAP阳性细胞。方差分析表明不同时间点的阳性细胞率存在的差别具有显著意义(F=702.93,P<0.05),LSDt检验表明除第7d与第9d无明显差别外,其余各时间点之间均有明显差别。其中第7天的分化率最高为87.60%±6.26%。结论本实验利用反义寡核苷酸序列特异性阻断了Hes1的表达,促进了神经干细胞向GABA神经元分化,并发现在诱导分化7d后分化率达到最高值。     

脂肪源性γ-氨基丁酸能神经元移植治疗帕金森病大鼠的疗效观察

目的 研究脂肪来源的间充质干细胞诱导为γ-氨基丁酸(GABA)能神经元的方法 ,探讨GABA能神经元移植治疗帕金森病模型大鼠的疗效. 方法 取大鼠脂肪组织.利用本单位自行配制的神经干细胞培养基诱导分化为神经干细胞,利用GABA能神经元定向诱导培养基对神经干细胞进行二次定向诱导,并对其进行特异性鉴定.将诱导成功的神经干细胞、GABA能神经元分别移植入帕金森病大鼠模型的丘脑底核,在移植后2周、4周、8周观察大鼠行为学变化情况.结果 体外扩增的脂肪间充质干细胞经过神经干细胞培养基培养后,细胞定向诱导并表达巢蛋白、神经元烯醇化酶(NSE)等神经干细胞标志.经GABA能神经元定向分化培养基二次诱导后,免疫荧光鉴定细胞GAD65阳性.立体定向移植细胞4周后,神经干细胞组与GABA能神经元组的大鼠行为学均得到改善,且GABA能神经元组的疗效更加显著. 结论 脂防来源的间充质干细胞可诱导分化为GABA能神经元,将其移植人大鼠的丘脑底核可以明显改善帕金森病大鼠的运动功能.     

大鼠胎脑神经干细胞的培养分化及鉴定的实验研究

目的建立大鼠胎脑皮质神经干细胞的培养、扩增、诱导分化及鉴定的方法。方法选取孕14d胎鼠的大脑皮质作为细胞来源,在无血清培养基中添加B27、碱性成纤维细胞因子、表皮生长因子,建立胚胎神经干细胞的体外培养体系,用5%的胎牛血清诱导神经干细胞分化,用免疫荧光染色技术进行对神经干细胞及诱导分化细胞的鉴定。结果原代培养的神经干细胞折光性强,培养第2d开始形成细胞集落,第3d形成大的神经球。3～5代传代的细胞生长稳定。经胎牛血清诱导后第3d开始,神经球开始向周边发出突起,悬浮的细胞开始贴壁生长。原代及传代培养的神经球表达Nestin阳性,分化细胞分别表达NSE、GFAP和GALC阳性。结论应用无血清培养技术可在体外培养、扩增出神经干细胞。5%的胎牛血清可以诱导神经干细胞向神经元、星形胶质细胞和少突胶质细胞等成熟细胞分化。     

脑源性神经营养因子对胎鼠神经干细胞向神经元方向分化的诱导

背景：神经元是中枢神经系统起主导功能的细胞，培养干细胞的最终目的是获得相应表型的神经元，从而促进中枢神经系统损伤的修复。但以往相关文献显示神经干细胞分化为神经元的比例不到30%。 目的：以脑源性神经营养因子作为诱导分化剂，观察其对胎鼠源性神经干细胞向神经元方向分化的作用。 设计、时间及地点：细胞学体外观察，于2006-06/2007-08在南京医科大学第一附属医院中心实验室完成。 材料：清洁级孕14~17 d的SD大鼠10只，诱导分化剂脑源性神经营养因子为Peprotech产品，胎牛血清为杭州四季青产品。 方法：孕鼠处死后取出胚胎，剪碎后机械法体外分离培养神经干细胞，锥虫蓝染色计数，调整细胞密度为2×106，添加B27及碱性成纤维细胞生长因子，置于37 ℃、体积分数为0.05的CO2恒温箱中原代培养，待细胞克隆球明显增大、中央变暗时传代扩增。将培养所得的干细胞克隆球分为2组，诱导组添加体积分数为0.01的胎牛血清+20 μg/L脑源性神经营养因子共培养，血清对照组仅添加体积分数为0.01的胎牛血清。 主要观察指标：在倒置显微镜下观察细胞生长、贴壁和分化情况。诱导分化5 d后，行神经元免疫组化鉴定，流式细胞仪检测神经元阳性细胞率。取原代培养未分化的神经干细胞及诱导分化3 d的两组细胞，RT-PCR检测内源性bHLH基因MASH-1的表达。 结果：原代培养可获得数十至数百个神经干细胞聚集在一起的神经克隆球，形态规则，立体感强；悬浮生长的克隆球经诱导分化后，逐渐失去其球状的立体外观，邻近克隆球发出的突起可相互连接，3 d后即有神经元、星形胶质细胞和少突胶质细胞从克隆周围出现。培养的神经干细胞呈巢蛋白阳性表达，诱导分化后呈微管相关蛋白2阳性表达。与血清对照组比较，诱导组神经元阳性细胞率显著升高（χ2=16.0，P < 0.05）。与未分化的神经干细胞比较，诱导组、血清对照组细胞MASH-1的表达均明显升高，且前者升高幅度明显强于后者（F=21.7，P < 0.05）。 结论：脑源性神经营养因子能促进神经干细胞向神经元方向分化，可能与内源性bHLH基因MASH-1的表达升高有关。     

星形胶质细胞源性因子对神经干细胞分化的实验研究

目的探讨星形胶质细胞源性因子对神经干细胞分化的影响。方法分离和培养新生大鼠脑组织的神经干细胞;采用差速贴壁法和振荡法分离纯化星形胶质细胞,用免疫细胞化学染色法,胶质纤维酸性蛋白(GFAP)标记星形胶质细胞,进行细胞的纯度鉴定;将星形胶质细胞和神经干细胞在互不接触的情况下进行共培养,免疫荧光法观察神经干细胞分化后神经元特异性烯醇化酶(NSE)、GFAP和酪氨酸羟化酶(TH)的表达。结果纯化的星形胶质细胞GFAP抗体标记阳性,细胞纯度达98%;星形胶质细胞与神经干细胞共培养时,神经干细胞贴壁分化加快,NSE阳性细胞及TH阳性细胞明显多于对照组(P<0·05)。结论星形胶质细胞源性因子可快速诱导神经干细胞向神经元细胞、包括多巴胺神经元细胞分化,提示星形胶质细胞支持神经元发生。     

人小龄胚胎神经干细胞的分离培养、扩增及鉴定

目的 探索人胚胎神经干细胞的体外分离培养条件，从而在体外大量扩增神经干细胞；并观察神经干细胞增殖、分化的特点。方法 从人胚胎脑分离神经干细胞，部分细胞冻存，另一部分细胞进行体外培养。采用无血清培养液，加入表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF)刺激细胞增殖，进行体外扩增、传代培养。采用免疫荧光法鉴定神经干细胞和分化的神经元及胶质细胞。结果 从人胚胎脑分离的细胞在含有EGF和bFGF的无血清培养液中能形成大量的神经干细胞球，这些神经干细胞球可在体外扩增及传代培养。免疫荧光法鉴定神经干细胞球中大部分为神经上皮干细胞蛋白(nestin)表达阳性细胞。贴壁后可以分化出神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)表达阳性的细胞。经冻存后的胎脑细胞也能培养出具有同样特征的神经干细胞。结论 在含有EGF和bFGF的无血清培养液中，从人胚胎脑能分离培养出神经干细胞，并能在体外大量扩增。这为人类神经干细胞的进一步研究和应用提供了材料。     

神经干细胞在创伤性脑损伤灶周边的成活和迁移*

背景：成年人中枢神经系统再生困难,颅脑损伤后,损伤灶周边区域神经细胞的存活数量直接影响患者的预后。如何有效地使移植入创伤性脑损伤灶周边的神经干细胞存活分化,是目前神经修复再生研究的重点。 目的：探讨神经干细胞移植入大鼠创伤性脑损伤灶周边的成活、迁移和分化情况。 方法：利用无血清培养技术,加入表皮生长因子、碱性成纤维生长因子诱导刺激大鼠胚胎源性前脑神经干细胞生长增殖,并在体外进行克隆培养,移植前行BrdU标记,采用免疫组化和免疫荧光法检测其增殖特性和多向分化潜能,并观察其移植到Fenney’s落体脑损伤模型鼠脑皮质内的成活和迁移情况。 结果与结论：免疫组化及免疫荧光检测结果显示克隆细胞球呈nestin和BrdU阳性,分化后呈NSE,GFAP,MAP-2阳性。免疫组化及荧光双标检测结果显示移植后7,14 d损伤灶周边散在BrdU阳性细胞,并且GFAP阳性细胞增多。提示前脑神经干细胞在体外培养中能够增殖,并分化为神经元和神经胶质细胞,移植后能够在创伤性脑损伤灶周边存活和迁移,形态上显示出与脑组织整合的特点。     

GAD65基因转染神经干细胞的实验研究

目的检测神经干细胞经谷氨酸脱羧酶（GAD65）基因转染并诱导分化后γ-氨基丁酸（GABA）的表达,为GAD65基因修饰神经干细胞移植治疗癫痫提供理论依据。方法采用新一代高效脂质体作为转染试剂的基因转导方法转染神经干细胞,通过免疫细胞荧光方法鉴定神经干细胞分化为GABA阳性神经元情况。结果采用脂质体转染方法能成功地将GAD65基因转染至神经干细胞中,转染的神经干细胞在分化第7天及第14天GABA阳性神经元分别占（31．9±4．2）％、（26．5±2．3）％,未转染的神经干细胞分化的GABA阳性神经元则分别占（28.4±1．9）％和（20．3±3．5）％,转染者与未转染者相比,分化的GABA阳性神经元百分比有显著性差异（P〈0．05）。结论采用脂质体基因转染技术,可以将GAD65基因转染至神经干细胞中,并促进神经干细胞分化为GABA神经元。     

大鼠脂肪基质细胞的分离培养及二步法诱导分化神经细胞

目的 研究脂肪基质细胞体外培养扩增及向神经细胞诱导分化的新方法.方法 取SD大鼠的腹膜后脂肪组织,以Ⅰ型胶原酶消化离心,含10%胎牛血清的DMEM/F12培养基重悬细胞,增殖传代,取第5代细胞进行二步法诱导分化.首先以无血清Neurobasal培养基联合应用碱性成纤维细胞生长因子(bFGF) 20 ng/mL、表皮生长因子(EGF)20 ng/mL和N2(1:100)添加剂将细胞先诱导成神经球;再取第二代神经球加入1%胎牛血清、5%马血清、0.5μmol/L的维甲酸(RA)和50ng/mL脑源性神经营养因子(BDNF),促进其向神经元和胶质细胞方向分化;免疫荧光染色鉴定细胞分化结果.结果 初接种的细胞大多呈圆球状,悬浮在培养基中,24 h后贴壁.随着细胞密度加大,形态变成单层成纤维样细胞,呈漩涡状排列.无血清Neurobasal培养基诱导5～7 d可见悬浮的神经球出现;神经球迅速增殖,14d时直径可达100μm.分化后的大部分细胞呈现典型的神经元样改变,细胞体为圆形,内可见明显的细胞核,细胞伸出细长突起或网状突起,可见较多的细胞形成网络连接.免疫荧光染色鉴定结果证实,除了神经球可以高表达巢蛋白(nestin)(阳性率75.62%±1.34%)之外,分化细胞还可有效表达β-mbulinⅢ(未成熟神经元标记物,阳性率达57.62%±4.92%)或微管相关蛋白2(MAP2ab)(成熟神经元标记物,阳性率11.25%±3.87%);有少量细胞表达胶质纤维酸性蛋白(GFAF)(阳性率18.34%±3.87%)和GalC(阳性率14.35%+3.98%).结论 从成年大鼠脂肪中分离得到的脂肪基质细胞可在体外培养得到大量分化潜能稳定的子代细胞.应用本实验二步诱导法,可得到较高比例的nestin及β-mbulinⅢ阳性细胞,并有一定比例MAP2ab阳性细胞出现.     

维甲酸诱导胚胎大鼠脑海马神经干细胞向神经元的分化

背景：目前认为直接进行神经干细胞移植后细胞虽能存活,但是只分化成神经胶质细胞,并不能分化成有功能的神经元。 目的：探讨维甲酸诱导对胚胎大鼠脑海马神经干细胞向神经元分化的作用。 设计、时间及地点：细胞学体外实验,于2008-09/2009-02在辽宁医学院科技实验楼完成。 材料：胚龄13.5 d的SD大鼠由辽宁医学院实验动物中心提供。 方法：分离胎鼠脑海马组织,胰蛋白酶消化法体外培养获得神经干细胞。将原代和传代细胞以1×107 L-1接种到培养孔中,分别进行常规贴壁分化培养和维甲酸诱导分化培养。 主要观察指标：神经干细胞的鉴定,光镜及免疫组化检测神经干细胞诱导分化结果。 结果：免疫组织化学检测结果显示,原代和传代后得到的神经干细胞团均呈巢蛋白阳性。常规贴壁分化培养7 d后,神经元多呈椭圆型和近似三角形,胞体大,细胞边缘清楚,胞体上有多个突起;而维甲酸诱导分化培养后,神经元数量增加,形态清楚,但细胞胞体上突起较少。与常规贴壁分化培养比较,维甲酸诱导分化培养后神经干细胞向神经元分化率明显升高(P < 0.01),神经干细胞向神经胶质细胞分化率明显降低(P < 0.01)。 结论：从大鼠胚胎脑海马组织中分离得到可自我复制和多向分化的神经干细胞,维甲酸体外诱导后可以增加其向神经元方向分化的比例。     

沙盘游戏疗法在地中海贫血患儿心理干预中的应用

本文目的是对沙盘游戏疗法在地中海贫血患儿心理干预中的应用进行综述,以期为地中海贫血患儿的心理康复提供参考。地中海贫血是以珠蛋白生成障碍为主要特征的遗传性疾病,由于长期输血治疗,患儿存在较多的心理和行为问题。沙盘游戏疗法作为一种有效、实用的儿童心理治疗方法,对提高地中海贫血患儿的康复效果、改善生存质量有重要的临床意义。     

癫痫共病抑郁的机制及临床诊疗

本文目的是探讨癫痫共病抑郁的可能机制及临床诊疗。癫痫是一种常见的、慢性的、致残性的神经疾病,癫痫患者生活质量下降,存在明显的负性情绪,常伴发各种精神疾病。癫痫与抑郁具有共同的神经生物学基础,可能存在共同的发病机制。本文从癫痫共病抑郁的发病机制、临床诊断及治疗方面予以总结归纳。     

Efficacy and Effectiveness of Child and Adolescent Psychotherapy and Pharmacotherapy

Decades of intervention research have produced a rich body of evidence on the effects of psychotherapies and pharmacotherapies with children and adolescents. Here we summarize and critique that evidence. We review findings bearing on the efficacy of psychosocial treatments and medications under controlled experimental conditions. We also report evidence, where available, on the effectiveness of both classes of treatment with clinically referred youth treated in real-world clinical contexts. In general, the large body of evidence on efficacy contrasts sharply with the small base of evidence on effectiveness. Addressing this gap through an enriched research agenda could contribute importantly to linking scientific inquiry and clinical practice—to the benefit of both ventures. This is one element of a multifaceted agenda for future research and for synthesis of research, which will require the interplay of multiple disciplines related to child and adolescent mental health.     

Cultural Identity and Experiences of Prejudice and Discrimination of Afghan and Iranian Immigrant Youth

In culturally diverse and immigrant receiving societies, immigrant youth can be subject to prejudice and discrimination. Such experiences can impact on immigrant youth’s cultural identity and influence their psychosocial outcomes. This paper presents findings of a study that examined cultural identity and experiences of prejudice and discrimination among Afghan (N = 9) and Iranian (N = 17) immigrant youth in Canada. The study had a prospective, comparative, longitudinal qualitative design. Data was gathered through focus groups, interviews, journals and field logs. Four main themes emerged on participants’ experiences of prejudice and discrimination: (a) societal factors influencing prejudice; (b) personal experiences of discrimination; (c) fear of disclosure and silenced cultural identity; and (d) resiliency and strength of cultural identity. Drawing from Rosenberg’s (Conceiving the self, Basic Books, New York, 1979) self-concept framework and Romero and Roberts (J. Adolesc., 21:641–656, 1998) distinction between prejudice and discrimination, results indicated that youth’s extant and presenting cultural identity were affected. Inclusive policies and practices are needed to promote youth integration in multicultural and immigrant receiving settings.

发作性睡病与异态睡眠的诊治

本文目的是探讨发作性睡病与异态睡眠的诊断与治疗.发作性睡病被漏诊和误诊的几率较高,危害较大,共患异态睡眠比例高.文章从发作性睡病临床特征、REM睡眠的作用、发作性睡病与异态睡眠(睡眠瘫痪、睡眠幻觉、快眼动睡眠期行为障碍)共病特征及治疗这四个方面进行了讨论.     

Effects of Agonists and Antagonists of Cholecystokinin on Contractile and Myoelectric Activity of Isolated Muscle of Colon and Ileum in the Dog and Guinea Pig

This study evaluates the effects of agonists and antagonists of cholecystokinin (CCK) on contractile and myoelectrical activity in isolated longitudinal muscle strips from colon or ileum of guinea pigs or beagle dogs. Caerulein and CCK-8 caused a dose-dependent increase of contractile and myoelectrical spike activity in both species with maximal effects seen between 10⁻⁸ and 3 × 10⁻⁸ M. The dose responses were identical for both CCK agonists and species. The dose-related effects of CCK compounds on colonic muscle were slightly shifted to the right when compared to ileum in both species. All antagonists, the proglumide-derivatives CR1409, CR1392, and CR1505, as well as the nonpeptide substances asperlicin and L-364,718, caused a parallel rightward shift of CCK's dose-dependent motor activity response, indicating the competitive nature of inhibition. The antagonists displayed a rank order of potency in antagonizing CCK's action on intestinal motility similar to their ability to antagonize CCK's action on pancreas and gallbladder. L-364,718 was the most potent antagonist, followed by CR1409, CR1505, CR1392, asperlicin, and proglumide. The antagonists did not affect contractile or myoelectrical responses to acetylcholine, histamine, motilin, or substance P. Thus compounds that have been described as CCK antagonists for pancreas and gallbladder also act as specific and competitive antagonists of CCK's action on contractile and myoelectrical activity of Heal and colonie muscle.     

小胶质细胞和少突胶质细胞前体的培养和鉴定

目的 探讨新生大鼠脑组织小胶质细胞(MG)和少突胶质细胞(OL)前体的分离和体外培养方法 . 方法 取新生2 d SD大鼠脑组织,体外原代培养混合胶质细胞7 d后,分别采用"改良振荡伴差速贴壁"法和"营养缺失伴振荡"法纯化培养MG和OL前体,并分别应用免疫荧光染色异凝集素-B4(IB4)和OL前体标记物(O4)进行鉴定.结果 混合胶质细胞培养7 d后呈明显三层增长,其中MG位于上层,星型胶质细胞位于底层,两者之间为2型少突星型(O2A)祖细胞.纯化培养后OL前体胞体呈小圆形,有双极或三极突起,MG则以阿米巴形、圆形居多,或边缘呈毛刺状.免疫荧光染色IB4显示绿色荧光,MG纯度达到90%以上.免疫荧光染色O4显示棕黄色荧光,OL前体纯度达到95%以上. 结论 采用"改良振荡伴差速贴壁"法以及"营养缺失伴振荡"法分别成功获取大量纯度高、活力好的MG和OL前体.     

The neuroendocrinology of stress and the pathophysiology and therapy of depression and anxiety

Clinical and preclinical studies have gathered substantial evidence that stress response alterations play a major role in the development of major depression, panic disorder, and post-traumatic stress disorder. The stress response, the hypothalamic pituitary adrenocortical (HPA) system and its modulation by corticotropin-releasing hormones (CRH),corticosteroids,and their receptors, and the roles of natriuretic peptides and neuroactive steroids are described. We review the role of the HPA system in major depression, panic disorder, and post-traumatic stress disorder and its possible relevance for treatment. Impaired glucocorticoid receptor function in major depression is associated with an excessive release of neurohormones such as CRH, to which a number of signs and symptoms characteristic of depression can be ascribed. In panic disorder, a role of central CRH in panic attacks has been suggested. Atrial natriuretic peptide (ANP) is causally involved in sodium lactate-induced panic attacks. Furthermore, preclinical and clinical data on its anxiolytic activity suggest that nonpeptidergic ANP receptor ligands may be potentially useful in the treatment of anxiety disorders. Post-traumatic stress disorder is characterized by a peripheral hyporesponsive HPA system and elevated CRH concentrations in the CSF. This dissociation is probably related to an increased risk of this disorder. We further review recent data that describe an important role of GABA(A)-receptor modulatory,3 alpha-reduced neuroactive steroids in major depression, anxiety, and its treatment. Antidepressants are effective in both depression and anxiety disorders and have major effects on the HPA system,especially on glucocorticoid and mineralocorticoid receptors. Normalization of HPA system abnormalities is a strong predictor of the clinical course, at least in major depression and panic disorder. Currently,CRH-R1 or glucocorticoid receptor antagonists and ANP receptor agonists are being studied and may provide future treatment options more closely related to the pathophysiology of these disorders.     

Review and meta-analysis of the phenomenology and clinical characteristics of mania in children and adolescents

OBJECTIVE: Using predetermined criteria for study quality and methods, a literature review and meta-analysis of seven reports about pediatric bipolar disorder (BPD) was conducted to determine if there is a consistent picture of the phenomenology and clinical characteristics of BPD in children and adolescents. METHODS: Searches were conducted in MedLine and PsycINFO using the terms mania, BPD, children and adolescents, and was limited to published articles in peer-reviewed journals. Seven reports were selected that met the following criteria: a systematic method for the elicitation and reporting of symptoms and clinical characteristics of subjects; subjects were interviewed by a trained researcher or clinician; ages 5-18 years; use of a diagnostic system, either DSM or RDC for categorization; a consensus method for the establishment of the diagnosis of BPD. RESULTS: Most DSM-IV symptoms of mania were common in the children and adolescents with BPD with the most common symptoms being increased energy, distractibility, and pressured speech. On average, four of five bipolar cases also showed threshold levels of irritable mood and grandiosity, and more than 70% of all cases showed elated/euphoric mood, decreased need for sleep, or racing thoughts. Roughly 69% of cases also showed poor judgment, whereas only half of bipolar cases demonstrated flight of ideas, and slightly more than one-third showed hypersexuality or psychotic features. CONCLUSIONS: The clinical picture that emerges is that of children or adolescents with periods of increased energy (mania or hypomania), accompanied by distractibility, pressured speech, irritability, grandiosity, racing thoughts, decreased need for sleep and euphoria/elation.