Laminin促进胚胎中脑多巴胺神经元生长发育的体外培养研究

本文在体外培养中,研究Laminin(LN)对大鼠胚胎中脑多巴胺(DA)神经元生长发育的影响。将胚胎鼠(胎龄15天)腹侧中脑细胞悬液,接种于底部涂有LN的24孔培养板中,培养2～6天后取出,以酪氨酸羟化酶(TH)免疫组织化学ABC法观察DA神经元的生长情况。培养48小时后,几乎所有TH免疫阳性细胞都有突起伸出,以双极细胞为主,有些突起分支末端有活跃生长的生长锥,后者又可发出多根丝状伪足。在培养3～6天的标本中,除见有单极、双极TH免疫阳性细胞外,每孔均见有     

纹状体对体外培养中脑多巴胺神经元形态发育的影响

靶细胞在神经细胞的生长发育过程中起着重要的调节作用。本文通过胚胎腹侧中脑和纹状体联合培养研究纹状体对中脑多巴胺(DA)神经元形态发育的影响。纹状体(Str)和腹侧中脑(YMA)细胞悬液取自胚胎14天SD大鼠,联合培养3到14天后取出,用酪氨酸羟化酶(TH)免疫组化ABC法观察不同时期DA神经元的生长。与VMA单独培养相比,联合培养3天,发现细胞聚集程度较低,开始形成单层分布,培养7天TH阳性细胞数增加,多突起阳性细胞较易发现,细胞突起粗短,呈树枝状分枝。培养14天,TH阳性细胞数进一步增加,平均可达49个/25mm~2。本文对纹状体调节DA神经元生长发育的机制进行了讨论。     

BDNF、SDNF、GM_1对体外培养的多巴胺神经元的影响

本研究在体外培养的条件下研究了BDNF、SDNF、GM1对大鼠胚腹侧中脑多巴胺神经元生长发育的影响。取孕15d鼠胚腹侧中脑细胞悬液将之接种于24孔培养板中进行培养。实验分4组；对照组及分别向培养液中加入BDNF、SDNF、GM1等的三个实验组。培养7d后取出。用酪氨酸羟化酶免疫组织化学ABC法观察和比较了多巴胺神经元的生长状态。发现加入BDNF、SDNF、GM1的培养孔内的酪氨酸羟化酶阳性神经元明显增多，细胞突起的长度增加且数量增多，与单纯培养组相比均有显著性差异，但三个实验组之间无显著性差异。研究结果表明，BDNF、SDNF、GM1可促进体外培养的鼠胚腹侧中脑多巴胺神经元的存活及突起的延伸。     

体外长期培养大鼠中脑多巴胺神经元的形态学观察

本文研究了中脑多巴胺神经元在体外长期培养过程中的形态发育。动物选用胚胎14天SD大鼠,取中脑腹侧部制成细胞密度为2×10~5/ml的悬液,接种于24孔培养板中,培养1天至6周,以酪胺酸羟化酶免疫组化方法、单纯荧光组化方法和外源性儿茶酚胺荧光组化方法,观察中脑多巴胺神经元的体外长期培养发育状况。培养3天后多巴胺神经元已有单极或双极的突起发出,培养1周突起进一步伸长,并在沿途开始发出分支,有些末端见有生长锥。除散在分布的多巴胶神经元外,还能见到多巴胺神经元细胞群。培养2至3周,突起的分支增多,并明显出现念珠状膨大。在培养4-6周的标本中,仍可见到部分发育成熟的多巴胺神经元。多巴胺神经元在体外培养中的形态可分为二类,即梭形细胞和多极细胞。棱形细胞一般有两个突起,分别从胞体两极发出;多极细胞胞体呈圆形、多角形或三角形,可发出3-6个突起。实验结果表明多巴胺神经元的体外发育过程与在体相似。     

单唾液酸神经节苷脂对MPTP损害中脑多巴胺神经元的保护作用

本文研究单唾液酸神经节苷脂（ＧＭ１）对大鼠中脑多巴胺（ＤＡ）神经元体外生存及其损伤后的保护作用。取胚胎腹侧中脑制成细胞悬液，常规体外培养。实验分为四组：第一组在培养液中加入ＧＭ１（ＧＭ１组）；第二组在培养过程中使用１－甲基－４－苯基－１，２，３，６－四氢吡啶（ＭＰＴＰ）破坏ＤＡ神经元（ＭＰＴＰ组）；第三组在使用ＭＰＴＰ破坏ＤＡ神经元的同时给予ＧＭ１（ＧＭ１－ＭＰＴＰ组）；第四组为正常对照组。四组培养细胞定期抽出作免疫组化和荧光组化染色。ＧＭ１组培养各阶段，酪氨酸羟化酶（ＴＨ）免疫反应阳性细胞数量均明显多于对照组，至体外培养２周时，每孔阳性细胞数平均可达７２．８个，与对照组相比有显著性差异。ＭＰＴＰ组在加入ＭＰＴＰ１周左右，荧光组化阳性细胞已基本消失，此后ＴＨ免疫反应阳性细胞逐渐减少，至体外培养２周，每孔ＴＨ阳性细胞数平均仅剩１４．５个，与对照组相比有显著性差异。在残存的ＤＡ神经元中，突起变短或消失。ＧＭ１－ＭＰＴＰ组，体外培养期间均可看到荧光组化阳性细胞，ＴＨ免疫反应阳性细胞数量在体外培养２周时平均每孔为３１．７个，明显高于ＭＰＴＰ组。研究结果表明ＧＭ１能提高体外生长的ＤＡ神经元的生存率，并能减轻ＭＰＴＰ对?     

纹状体和中缝核外植块促进体外培养的中脑多巴胺神经元的发育

为了探讨神经元与其靶组织之间的相互作用及作用因素，建立了不同时期的纹状体外植场与１４ｄ大鼠胚胎中脑黑质神经元联合培养；１４ｄ大鼠胚胎中缝核外植块与黑质神经元联合培养。结果表明，生后２周、新生及１４ｄ大鼠胚胎纹状体外植块和１４ｄ大鼠胚胎中缝核外植块，对中脑黑质多巴胺神经元的形态发育有明显促进作用，其中包括长突起神经元数目和神经元突起长度的增加。上述３个时期的纹状体外植块，对中脑黑质多巴胺神经元的作用     

中药颤复宁对多巴胺神经元的保护作用及其机理

目的:观察中药颤复宁血清对多巴胺神经元的生长状况和细胞内钙离子浓度的影响,并探讨其保护作用的机理。方法:用血清药理学方法制备中药颤复宁大鼠血清,加入到6-羟基多巴(6-OHDA)处理的多巴胺神经元培养液中,检测酪氨酸羟化酶(TH)阳性细胞数及测量TH阳性神经元胞体面积和最长突起的长度。用激光扫描共聚焦显微镜检测细胞内钙离子浓度。结果:6-OHDA损伤后,对照血清组多巴胺神经元数量减少,突起变短,细胞内钙离子浓度升高。而颤复宁血清组TH阳性细胞数量多,多巴胺神经元最大突起长度长,细胞内钙离子浓度明显降低(P<0.01)。结论:中药颤复宁血清能提高多巴胺神经元的生长活性,平衡细胞内钙离子浓度,对6-OHDA体外毁损的多巴胺神经元具有直接的保护作用。     

不同时程吗啡依赖对大鼠中脑腹侧被盖区多巴胺能神经元的影响

目的:观察不同时程吗啡依赖对大鼠中脑腹侧被盖区(ventral tegmental area,VTA)多巴胺能神经元的影响。方法:建立吗啡慢性依赖大鼠模型,石蜡包埋组织连续切片,免疫组织化学染色观察多巴胺能神经元特异性标记物酪氨酸羟化酶(tyrosine hydroxylase,TH)的表达变化。结果:TH免疫组化结果显示随着吗啡依赖时间的延长VTA区内TH阳性细胞逐渐减少,吗啡依赖6周组阳性细胞减少明显。结论:较长时程吗啡依赖大鼠VTA多巴胺能神经元损伤明显。     

Sertoli细胞诱导大鼠胚胎中脑神经干细胞定向分化的研究

目的研究Sertoli细胞对胚胎中脑神经干细胞的营养及向多巴胺能神经元分化的诱导作用。方法将大鼠Sertoli细胞与胚胎13d大鼠中脑神经前体细胞体外共培养5、7、10、14d后,免疫荧光检测神经干细胞标记物Nestin、神经元前体细胞标记物β-Ⅲ tubulin、多巴胺能神经元标记物酪氨酸羟化酶(TH)的表达情况。结果随着共培养时间的延长,神经干细胞中nestin阳性细胞数量逐渐减少,β-Ⅲ tubulin阳性细胞数在共培养7d时较多,TH阳性的神经元在共培养10d时数量最多。而且在各时间段TH阳性细胞数均多于单独培养的神经前体细胞。结论Sertoli细胞能够诱导与其共培养的胚胎中脑神经干细胞定向分化为多巴胺能神经元。     

谷氨酸、N-甲基-D-天冬氨酸和海人藻酸在体外对大鼠中脑腹侧神经元的影响

本研究分别以谷氨酸、N-甲基 -D-天冬氨酸和海人藻酸作用于体外培养的新生大鼠中脑腹侧神经元 ,经抗酪氨酸羟化酶和抗γ-氨基丁酸双重免疫荧光染色 ,观察了谷氨酸等三者对不同神经元的毒性作用。结果表明 ,大鼠中脑腹侧部主要含有多巴胺和 γ-氨基丁酸神经元 ,多巴胺神经元对谷氨酸等三者的毒性作用均较 γ-氨基丁酸神经元敏感。多巴胺能神经元对谷氨酸毒性的脆性 ,在培养的第一周主要由海人藻酸受体介导 ,以后则以 N-甲基 -D-天冬氨酸受体为主。     

Specific effects of platelet derived growth factor (PDGF) on fetal rat and human dopaminergic neurons in vitro

The neurotrophic effects of the BB isoform of platelet-derived growth factor (PDGF) on rat and human fetal mesencephalic dopaminergic neurons have been characterized in vitro. A dose-response analysis demonstrated maximal responses at 30 ng/ml of PDGF-BB. This concentration resulted in a marked increase in the survival and neurite outgrowth from rat and human tyrosine hydroxylase-(TH) positive, presumed dopaminergic neurons after 7 days in vitro. The effects of PDGF-BB on survival of TH-positive neurons were comparable to those of brain-derived neurotrophic factor (BDNF), whereas neurite outgrowth was more pronounced after addition of BDNF. The combination of BDNF and PDGF-BB yielded no additive effects. Double immunohistochemical staining of rat cultures demonstrated PDGF -receptors on about 90% of the TH-positive neurons. PDGF-BB treatment of rat mesencephalic cultures induced an upregulation of c-fos and TH mRNA with maximal levels after 0.5–2 h as assessed by quantitative PCR analysis. An increased number of Fos protein-positive cells was detected immunohistochemically after 4 h of PDGF-BB treatment. The present results provide further evidence for specific and direct effects of PDGF-BB on gene expression, survival and neurite outgrowth of mesencephalic dopaminergic neurons of rat and human origin.     

大鼠腹侧中脑多巴胺能神经元的形态学发育与分布特征

目的:探讨大鼠腹侧中脑多巴胺能(mDA)神经元的形态学发育和分布特征。方法:取胚胎晚期至成年大鼠不同时期的腹侧中脑连续冠状切片,以酪氨酸羟化酶(TH)免疫荧光染色方法观察mDA神经元。结果:(1)在胚胎期,TH阳性细胞多为圆形或椭圆形,随着发育进程,其突起逐渐伸长,分支也逐渐增多;出生后28 d,TH阳性细胞表现为成熟mDA神经元的形态特征。(2)Map-2/TH免疫荧光检测显示,新生期大鼠腹侧中脑TH阳性神经元定位分布与成年期的mDA神经元分布趋向一致。(3)E16.5 d时腹侧中脑TH阳性细胞的数量和密度最大,此后逐渐下降。结论:大鼠mDA神经元的发育在出生前后,呈现先大量形成后又逐渐减少的过程,与此同时其形态趋向成熟;至P0 d时mDA神经元分布定位基本完成,至出生后28 d形态学发育成熟。     

己烯雌酚对体外培养中脑神经元的保护作用

为观察己烯雌酚对体外培养的胎鼠中脑神经元的影响,将孕14d的SD胎鼠中脑取出,制成细胞悬液后种植在24孔培养板中。实验分空白对照组和己烯雌酚组(浓度分别为10-9、10-8、10-7mol/L)。用倒置显微镜观察各组细胞的存活及生长状况,并用免疫细胞化学方法测定各组细胞神经元特异烯醇化酶(NSE)及c-Fos蛋白阳性表达情况。结果显示,己烯雌酚浓度在10-8mol/L时细胞容易贴壁,突起形成早而多,NSE、c-Fos阳性细胞数与对照组及低浓度组有显著差异(P<0.05),与高浓度组无显著差异。由此提示己烯雌酚对中脑神经元具有保护作用,并能提高其功能活性。     

脑源性神经营养因子体外诱导神经干细胞向神经元分化

目的观察脑源性神经营养因子(BDNF)对新生SD大鼠海马神经干细胞在体外分化为神经元的作用。方法取新生SD大鼠海马组织,以无血清培养技术培养获得神经干细胞,在BDNF诱导下让其在体外分化,7d后,通过免疫荧光技术结合图像分析技术来观察分化所得神经丝(neurofilament,NF)抗原阳性神经元的比率及其最长突起的长度。结果BDNF组分化所得细胞NF阳性率为13.66%,神经元突起长度为(146.27±26.30)μm,对照组分化所得细胞NF阳性率为9.38%,神经元突起长度为(117.00±23.98)μm,两组结果有显著性差异。结论BDNF能提高新生SD大鼠海马神经干细胞体外定向分化为神经元的比率,并且能刺激新生神经元突起的生长。     

Platelet-derived growth factor promotes survival of rat and human mesencephalic dopaminergic neurons in culture

Summary The effect of two isoforms of platelet-derived growth factor (PDGF), PDGF-AA and PDGF-BB, was tested on dissociated cell cultures of ventral mesencephalon from rat and human embryos. PDGF-BB but not PDGF-AA reduced the progressive loss of tyrosine hydroxylase- (TH)-positive neurons in rat and human cell cultures. The mean number of TH-positive cells in the PDGF-BB-treated rat culture was 64% and 106% higher than in the control cultures after 7 and 10 days in vitro, respectively. Corresponding figures for human TH-positive neurons were 90% and 145%. The influence of PDGF-BB was specific for TH-positive neurons and not a general trophic effect, since no change of either total cell number or metabolic activity was found. In PDGF-BB-treated cultures of human but not rat tissue the TH-positive neurons had longer neurites than observed in control or PDGF-AA-treated cultures. These data indicate that PDGF-BB may act as a trophic factor for mesencephalic dopaminergic neurons and suggest that administration of PDGF-BB could ameliorate degeneration and possibly promote axonal sprouting of these neurons in vivo.     

Interleukin-6 as a neurotrophic factor for promoting the survival of cultured catecholaminergic neurons in a chemically defined medium from fetal and postnatal rat midbrains

Interleukin-6 (IL-6, human recombinant) promoted the survival of catecholaminergic neurons from fetal and postnatal rat midbrains as assessed by an immunohistochemical staining for tyrosine hydroxylase (TH) in culture using a chemically defined medium. The maximal dose of IL-6 for the cell survival of postnatal P15 rat mesencephalic TH-positive neurons in culture for 7 days was 50 ng/ml. The survival-promoting effects on P15 cultures were observed both in high- and low-density cultures. The survival effect of IL-6 on the cultured P15 TH-positive neurons was significant for only 4-15 days in vitro. However, the viable number of TH-positive neurons with IL-6 was less than that of the control at early points in the culture process (1-2 days in vitro). Continuous presentation of IL-6 was required for promoting survival. The optimal dose of IL-6 for the survival of fetal E16 midbrain TH-positive neurons was 5 ng/ml, and the survival promoting effect was less than that for the P15 cultures. The maximal dose of IL-6 for the survival of P2 TH-positive neurons was 5 ng/ml and that of P8 was 50 ng/ml, indicating that the response of rat mesencephalic TH-positive neurons to IL-6 changes during the first postnatal week.