NCAM介导GDNF保护多巴胺能神经元作用的研究

目的研究神经细胞黏附分子(neural cell adhesion molecule,NCAM)在胶质细胞系源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)保护帕金森(Parkinson's disease,PD)模型大鼠受损多巴胺(dopamine,DA)能神经元中的作用。方法SD大鼠右侧纹状体内立体定位注射6-羟多巴胺(6-OHDA)制备早期PD模型,而后分为4组:对照组(同侧黑质内注射PBS),NCAM组(同侧黑质内仅注射anti-NCAM抗体),GDNF组(同侧黑质内注射GDNF),NCAM阻断组(同侧黑质内注射anti-NCAM抗体30min后注射GDNF),采用免疫组织化学染色技术和免疫印迹技术,观察各组酪氨酸羟化酶(tyrosine hydroxylase,TH)的表达变化。结果GDNF组黑质致密部TH阳性神经元数目(92.44±16.96)及表达的量(44731.50±9765.30)明显多于对照组(56.83±14.27;22218.75±5925.39),差别有统计学意义(P<0.05);NCAM阻断组与GDNF组相比,该处TH阳性神经元数目及表达的量明显减少(NCAM阻断组:67.57±12.71,26891.00±6848.87;GDNF组:92.44±16.96,44731.50±9765.30),差别有统计学意义(P<0.05)。结论NCAM参与了GDNF保护DA能神经元的作用。     

神经细胞黏附分子在正常老化大鼠黑质致密部多巴胺能神经细胞的表达

目的：观察神经细胞黏附分子（NCAM）和酪氨酸羟化酶（TH）在大鼠黑质致密部（SNc）多巴胺（DA）能神经细胞表达的变化,探讨其发生机制。方法：健康雄性SD大鼠24只,分为成年组（4—5月龄）和老年组（≥24月龄）,取中脑黑质,分别进行TH和NCAM的免疫组织化学染色及免疫印迹检测蛋白表达,显微镜下计数免疫组化染色阳性神经细胞,灰度分析电泳条带。结果：SNc的DA能神经细胞几乎都表达NCAM,老年大鼠SNcTH阳性细胞总数及蛋白量均无减少（P〉0．05）,但尾侧段TH阳性细胞减少（P〈0．05）;NCAM在阳性细胞总数及蛋白量均减少（P〈0．05）,但各段阳性细胞的减少无统计学意义（P〉0．05）。结论：正常老化后大鼠的SNcDA能神经细胞DA合成降低,并且NCAM可能参与了这种DA的合成下降。     

GDNF基因修饰的骨髓基质干细胞分泌物对多巴胺能神经元的营养作用

目的:克隆GDNF基因并修饰骨髓基质干细胞,观察该工程细胞分泌物对多巴胺能神经元的营养作用。方法:应用逆转录聚合酶链反应(RT-PCR)方法从新生小鼠大脑皮层细胞克隆出GDNFcDNA片断,以pECPP-Cl为载体导入骨髓基质干细胞,制备稳定表达GDNF基因的MSCs工程细胞,收集并浓缩MSCs和GDNF’基因修饰的MSCs工程细胞的条件培养液,通过MTT、倒置显微镜和免疫组织化学的方法观察MSCs和GDNF基因修饰的MSCs工程细胞分泌物对多巴胺能神经元的营养作用。结果:MSCs和GDNF基因修饰的MSC工程细胞分泌物均能促进多巴胺能神经元的存活和生长,MSCs工程细胞作用更强。结论:成功构建了GDNF基因修饰的MSCs工程细胞,该细胞对多巴胺能神经元有明显营养保护作用,在帕金森病治疗中可能有重要价值。     

脑源性神经营养因子对帕金森病大鼠多巴胺能神经元的影响

目的观察脑源性神经营养因子对帕金森病(Parkinson’s disease,PD)大鼠模型黑质多巴胺能神经元的影响。方法选用Wistar种系大白鼠30只,体质量230~250g,随机分3组,通过左侧中脑黑质立体定向注射法,组1为生理盐水对照组(简称对照组)10只,注射相应量(5μL)的生理盐水;组2为注射6-OHDA制作帕金森病模型组(简称6-OHDA组)10只,注射6-OHDA,5μL(2μg/μL);组3为(6-OHDA+BDNF)组,在制成帕金森病模型后再向同侧中脑黑质注射BDNF 5μL(3μg/5μL),连续6d,1次/d。分别观察动物的旋转行为,免疫组化染色方法观察黑质酪氨酸羟化酶(tyrosine hydroxylase,TH)阳性神经元的数量,高效液相法测定纹状体部多巴胺(dopamine,DA)含量的变化。结果单侧黑质内注入6-OHDA制成帕金森病大鼠模型后,6-OHDA组与对照组比较,产生旋转行为,(6-OHDA+BDNF)组在观察旋转行为时,症状明显改善;镜下见TH阳性神经元主要见于对照组的黑质致密部,数量为(42.3±7.56)个/μm2,模型组黑质致密部TH阳性神经元数明显减少为(2.41±1.07)个/μm2,(6-OHDA+BDNF)组黑质致密部TH阳性神经元数为(15.36+3.04)个/μm2;纹状体部多巴胺含量:生理盐水组为(11.4±1.2)μg/g,6-OHDA组(3.6±0.5)μg/g,(6-OHDA+BDNF)组(5.5±0.6)μg/g。结论 BDNF能改善6-OHDA所致的帕金森病大鼠黑质多巴胺能神经元数目的减少;明显抑制6-OHDA引起的纹状体部多巴胺含量降低;并可抑制6-OHDA对黑质多巴胺能神经元的毒性作用。     

脑源性神经营养因子对帕金森病大鼠黑质多巴胺能神经元的影响

目的 观察脑源性神经营养因子（Ｂｒａｉｎ ｄｅｒｉｖｅｄ－ｎｅｕｒｏｔｒｏｐｈｉｃ ｆａｃｔｏｒ,ＢＤＮＦ）对帕金森病（Ｐａｒｋｉｎｓｏｎｄｉｓｅａｓｅ,ＰＤ）大鼠模型黑质多巴胺能神经元的影响。方法将６－羟基多巴胺用立体定向法注入大鼠一侧中脑黑质制作 ＰＤ大鼠模型,并在注入６－羟基多巴胺之间每天向预伤侧壳核区定位注入ＢＤＮＦ,采用酪氨酸羟化酶免疫组化方法及透射电镜等观察ＢＤＮＦ对黑质多巴胺能神经元的显微超微结构影响。结果　ＢＤＮＦ能改善６－羟基多巴胺造成的ＰＤ大鼠黑质多巴胺能神经元数目减少及超微结构影响。结论　ＢＤＮＦ可减轻６－羟基多巴胺对黑质多巴胺能神经元的毒性作用。     

稳定表达GDNF基因的骨髓基质干细胞对多巴胺能神经元的作用

目的 制备胶质细胞源性神经生长因子(GDNF)基因修饰的骨髓基质干细胞(MSCs)，观察其对多巴胺能神经元的作用，探索治疗帕金森氏病的新途径。方法 应用逆转录聚合酶链反应(RT-PCR)方法从新生小鼠大脑皮层细胞克隆出GDNF cDNA片断，以pEGFP-C1为载体导入MSCs，制备稳定表达GDNF基因的MSCs工程细胞，采用联合培养的技术通过倒置显微镜和免疫组织化学的方法观察MSCs和GDNF基因修饰的MSCs工程细胞与多巴胺能神经元之间的相互作用。结果 MSCs和GDNF基因修饰的MSCs工程细胞均能促进多巴胺能神经元的存活和生长，MSCs工程细胞作用更强。结论 成功构建了GDNF基因修饰的MSCs工程细胞，该细胞对多巴胺能神经元有明显营养保护作用，在帕金森病治疗中可能有重要价值。     

稳定表达GDNF／EGFP融合基因的骨髓基质干细胞对多巴胺能神经元的保护作用

目的　 构建GDNF基因修饰的骨髓基质干细胞 ,并观察其对多巴胺能神经元的营养支持作用。方法　 应用逆转录聚合酶链反应 (RT PCR)方法从新生小鼠大脑皮层细胞克隆出GDNFcDNA片段 ,以pEGFP C1为载体导入骨髓基质干细胞 (MSCs) ,制备稳定表达GDNF/EGFP融合基因的MSCs工程细胞 ,用联合培养的技术通过倒置显微镜和免疫组织化学的方法观察MSCs和GDNF基因修饰的MSCs工程细胞与多巴胺能神经元的相互作用。 结果　MSCs和GDNF基因修饰的MSCs工程细胞共培养均能促进多巴胺能神经元的存活和生长 ,MSCs工程细胞作用更强。 结论　 成功构建了GDNF基因修饰的MSCs工程细胞 ,该细胞对多巴胺能神经元有明显营养保护作用 ,在帕金森病治疗中可能有重要价值     

神经细胞黏附分子结构及其在神经系统的作用

神经细胞黏附分子(NCAM)是一种主要表达于神经系统的细胞表面糖蛋白，具有典型的免疫球蛋白超家族的特征，以其Ig结构域所引发的特有的嗜同性和嗜异性介导细胞与细胞、细胞与胞外基质的相互作用，进而参与神经系统的发育过程，并发挥不可替代的作用。     

尼古丁对帕金森病大鼠纹状体GDNF和多巴胺含量的影响

目的 研究尼古丁对帕金森病（PD）大鼠纹状体脑胶质细胞源性神经营养因子(GDNF)和多巴胺（DA）含量的影响。方法 将6－羟多巴胺（6－OHDA）立体定向注射到大鼠右侧中脑腹侧背盖部（VTA）和黑质致密部（SNpc)，建立PD大鼠模型。采用生化、免疫组织化学方法观察不同剂量尼古丁对PD大鼠的作用，检测纹状体GDNF表达及DA含量的变化。结果 造模前及造模后皮下注射尼古丁的PD大鼠，纹状体GDNF表达及DA含量较PD组有明显改善（P＜0．05）。结论 尼古丁可减轻6－OHDA对黑质DA能神经元的损伤，对PD大鼠具有保护作用。     

首发精神分裂症患者疗效及血清多聚唾液酸神经细胞黏附分子、脑源性神经营养因子水平及利培酮的影响

目的:探讨首发未服药精神分裂症患者血清脑源性神经营养因子(BDNF)、多聚唾液酸神经细胞黏附分子(PSA-NCAM)水平及利培酮治疗对其的影响。方法:对62例首发精神分裂症患者(患者组)给予利培酮单一药物治疗6个月;采用酶联免疫吸附技术(ELISA)分别检测患者治疗前、治疗1个月及6个月时血清BDNF、PSA-NCAM水平,并与44例患者的正常一级亲属(亲属组)及38位健康志愿者(对照组)比较。结果:治疗前患者组血清BDNF水平明显低于对照组和亲属组,血清PSA-NCAM水平明显高于对照组和亲属组(P均0.05);对照组与亲属组间比较差异无统计学意义。治疗后,患者组血清BDNF水平逐渐升高,PSA-NCAM水平逐渐下降;与治疗前比较,治疗6个月时差异有统计学意义(P均0.05)。结论:首发未服药精神分裂症患者血清BDNF、 PSA-NCAM水平异常;利培酮治疗可改善血清BDNF及PSA-NCAM表达异常。     

GDNF partially protects grafted fetal dopaminergic neurons against 6-hydroxydopamine neurotoxicity

Rats were given unilateral 6-hydroxydopamine (6-OHDA) lesions and subsequently received transplants of fetal ventral mesencephalic tissue into the denervated striatum. Four weeks later transplanted animals were tested for graft-mediated reduction of amphetamine-induced rotational behavior. Subsequently, transplanted animals received an intrastriatal injection of either GDNF (10 microg) or citrate buffer into a site lateral to the transplant, and then 6 h later received an injection of either 4.0 microg of 6-OHDA, 8.0 microg of 6-OHDA, or vehicle using the same stereotaxic coordinates that were used for the GDNF/citrate buffer injection. Animals were re-tested for amphetamine-induced rotational behavior 2 weeks later. Histological analysis revealed a significant reduction in the number of cell bodies immunostained for tyrosine hydroxylase (TH+) within the transplant for those animals pretreated with an intrastriatal injection of citrate buffer and subsequently given either dose of 6-OHDA. Transplanted animals pretreated with GDNF and subsequently administered 8.0 microg of 6-OHDA showed a significant reduction of TH+ neurons within the transplant compared to controls, however TH+ cell counts for this group remained significantly higher than the TH+ cell counts for the group of animals receiving the same dose of 6-OHDA but pretreated with citrate buffer. GDNF pretreatment completely protected TH+ cell bodies against 4.0 microg of 6-OHDA. Rotational scores indicated that GDNF provided only partial protection against 6-OHDA neurotoxicity in terms of transplant function. For both groups of transplanted animals receiving GDNF pretreatment and 6-OHDA injections, amphetamine-induced rotational scores dropped below the scores for animals pretreated with citrate buffer but remained significantly higher than the scores for transplanted animals that were not injected with 6-OHDA. Both histological and behavioral measures indicate GDNF partially protects integrated transplants against neurotoxic insult.     

Neuroprotective effects of GDNF against 6-OHDA in young and aged rats

In young adult rats, glial cell line-derived neurotrophic factor (GDNF) can completely protect against 6-hydroxydopamine-induced loss of nigral dopamine neurons when administered 6 h prior to the 6-hydroxydopamine. The present study was undertaken to determine if GDNF would provide similar protective effects in aged rats. Male, Fischer 344 x Brown Norway hybrid rats of 3, 18 and 24 months of age were given an intranigral injection of GDNF or vehicle followed 6 h later with an intranigral injection of 6-hydroxydopamine. Nigral dopamine neuron cell survival, and striatal and nigral dopamine and DOPAC levels, were evaluated 2 weeks after the lesions. In vehicle treated animals cell survival on the lesioned side ranged from 15 to 27%. GDNF promoted significant cell survival in the nigra of all three age groups; however, the percent survival was lowest in the 24-month-old animals (85% at 3 months, 75% at 18 months, 56% at 24 months). Similarly, dopamine levels in the striatum and substantia nigra on the lesioned side remained significantly greater in the GDNF treated animals compared to the vehicle treated animals. As with the cell survival experiment, the protective effects of GDNF on dopamine levels were less in the 24-month-old animals. GDNF pretreatment also protected against 6-hydroxydopamine-induced reductions in striatal DOPAC levels in all age groups. Overall, these results indicate that GDNF can protect nigrostriatal dopamine neurons against the effects of 6-hydroxydopamine in aged as well as young adult rats. However, the extent of protection is less in the aged (24-month-old) animals.     

Activation of cell-survival signal Akt by GDNF in normal rat brain

Glial cell line-derived neurotrophic factor (GDNF) activates protein kinase Akt/PKB by phosphorylation (p-Akt) which plays key roles in cell survival. In the current study, we investigated a temporal expression of p-Akt by immunohistochemical analysis after a topical application of GDNF to normal cerebral hemisphere of rats. Although p-Akt immunoreactivity was weakly present in the sham control neural cells, GDNF application greatly enhanced it at 3 h, which lasted until 1 day. These results indicate that p-Akt is expressed in neuronal cells under physiological conditions, and that topical application of GDNF greatly enhanced the phosphorylation of Akt in normal rat brain.     

Association of the GDNF gene with depression and heroin dependence,but not schizophrenia,in a Chinese population

The association of seven GDNF tag SNPs with depression, heroin dependence (HD) and schizophrenia was evaluated in Chinese. An increased risk of HD and depression was associated with rs2910709 T/T genotype and rs884344 C allele, respectively, suggesting GDNF is a novel susceptibility gene for depression and HD.     

重组大鼠质粒pEGFP-GDNF的构建及大鼠胚胎神经干细胞转染的研究

目的 探讨携带GDNF基因的神经干细胞表达载体的构建方法。方法 采用RT-PCR方法从大鼠胎脑组织总RNA中扩增出该基因的全序列cDNA,并克隆到增强型绿色荧光蛋白（EGFP）报告基因的真核表达载体pEGFP-C1中,经酶切鉴定及测序分析对重组质粒pEGFP-GDNF作进一步鉴定。采用阳离子脂质体将重组质粒pEGFP-GDNF转染至鼠胚胎神经干细胞。结果 大鼠GD-NF cDNA已正确地克隆到真核表达载体pEGFP-C1中,而构建成重组大鼠质粒pEGFP-GDNF,GDNF基因在细胞中可稳定表达。结论 神经干细胞可直接作为基因靶细胞,能被GDNF真核细胞表达载体pEGFP-GDNF有效的感染。     

Cross-linking of NCAM receptors on neurons induces programmed cell death

Programmed cell death has been implicated in the loss of neurons that occurs in many neurodegenerative diseases. This has led to an increased interest in the types of stimuli that can initiate neurons to undergo programmed cell death. Previously, we have shown that cross-linking of membrane receptors with the lectin concanavalin A can trigger programmed cell death in neurons [D.H. Cribbs, V.M. Kreng, A.J. Anderson, C.W. Cotman, Cross-linking of Concanavalin A receptors on cortical neurons induces programmed cell death, Neuroscience 75 (1996) 173–185]. Concanavalin A, however, binds to many surface glycoproteins and therefore, it is important to determine whether certain specific receptors can initiate the program. We found that surface immobilized anti-neural cell adhesion molecules (NCAM) monoclonal antibodies provide a good substrate for adhesion and neurite outgrowth for cortical neurons. However, neurons treated directly with soluble anti-NCAM monoclonal antibodies show significant cell death after 24 h and exhibit the morphological and biochemical features indicative of apoptosis, including membrane blebbing, cell shrinkage, condensation of nuclear chromatin and internucleosomal DNA cleavage.     

抗坏血酸联合胶质细胞源性神经营养因子诱导神经干细胞向多巴胺能神经元分化的研究

目的 研究抗坏血酸(AA)和胶质细胞源性神经营养因子(GDNF)对神经干细胞向多巴胺能神经元分化的影响.方法 从新生24h内的sD大鼠脑组织分离和培养神经干细胞,进行神经干细胞鉴定.第二代神经干细胞诱导培养基中分别给予AA或(和)GDNF,10d后终止诱导,进行DA能神经元特异性标记物酪氨酸羟化酶(TH)和多巴胺转运蛋白的免疫细胞化学检测和TH基因的RT-PCR检测.结果 各诱导组均检测到TH mRNA的表达;与对照组比较,AA及GDNF均能增加NSC向TH阳性细胞分化的比率(P<0.05);与单独运用100μmol/LAA或10ng/mlGDNF组比较,联合诱导组可明显提高NSCs向TH阳性细胞分化的比率(P<0.05).结论 AA和GDNF均能促进NSCs向DA能神经元分化,两者联合诱导后分化作用得到进一步加强.     

尼莫地平对大鼠面神经损伤的保护作用及GDNF表达的影响

目的 探讨尼莫地平对大鼠面神经损伤的保护作用及对胶质细胞源性神经生长因子(GDNF)表达的影响.方法 96只大鼠按随机数字表法分为假手术组、单纯损伤组、尼莫地平预处理组、尼莫地平后处理组,后三组建立大鼠面神经电损伤模型.应用HE染色、Western blotting等方法,观察大鼠面神经损伤后不同时期(1、3、6月)GDNF的动态表达变化及尼莫地平不同给药时间对其表达的影响.结果 与单纯损伤组相比,尼莫地平预处理组大鼠面神经损伤程度减轻,GDNF表达升高,持续时间延长;尼莫地平预处理组GDNF表达和尼莫地平后处理组间比较差异有统计学意义(P＜0.05);尼莫地平后处理组GDNF表达在3月、6月时与单纯损伤组比较差异无统计学意义(P＞0.05).结论 预防性应用尼莫地平可以保护面神经,其机制可能是通过调节GDNF的表达而实现的.

Abstract：

Objective To study the protective function of nimodipine on facial nerve injury and its effect on the expression of glial cell line-derived neurotrophic factor (GDNF). Methods Ninety-six SD rats were randomly divided into sham-operated group, facial nerve injury group, nimodipine pretreatment group, and nimodipine post-treatment group. Rat models of facial nerve injury in thc later 3groups were established. The dynamic changes of expression of GDNF were observed by HE staining and Western blotting in different treatment groups and at different time points (1, 3 and 6 months after the injury). Restdts Compared with the facial nerve injury group, the nimodipine pretreatment and post-treatment groups had significantly less severe nerve damage and significantly up-rcgulated expression of GDNF (P＜0.05). The expression of GDNF in the nimodipine pretreatment group was statistically higher than that in the nimodipine post-treatment group (P＜0.05). However, the expression of GDNF in the nimodipine post-treatment group was not statistically different from that in the facial nerve injury group 3 and 6 months after the injury (P＞0.05). Conclusion Nimodipine has significant facial nerve protective effect, and one of the mechanisms of nimodipine to protect the facial nerve is to regulate the GDNF expression.     

The involvement of NF-kappaB p65/p52 in the effects of GDNF on DA neurons in early PD rats

Glial cell line-derived neurotrophic factor (GDNF) can exert neuroprotective effects on the substantia nigra pars compacta (SNc) dopaminergic (DA) neurons that are undergoing degeneration in Parkinson's disease (PD). In an attempt to investigate the molecular signaling mechanisms underlying GDNF protection the DA neurons from degeneration, we established early PD rat models in which the DA neurons in SNc were degenerating. Whether the cytoplasmic NF-κB signaling pathway was involved in the protection of GDNF on the degenerating DA neurons was examined in the present study. The results showed that the nuclear NF-κB p65 levels in the DA neurons increased when GDNF was injected into SNc of early PD rat models. Immunoprecipitation assays showed that the nuclear NF-κB p65/p52 complex levels increased after GDNF administration, while the p65/p50 complex levels decreased. These results indicated that GDNF could activate the NF-κB signaling pathway in the degenerating DA neurons. And it was the noncanonical NF-κB signaling pathway, which contained the NF-κB p65/p52 complex that was involved in the effects of GDNF on DA neurons.