Drug-delivering nerve conduit improves regeneration in a critical-sized gap

Autologous nerve grafts are the current “gold standard” for repairing large nerve gaps. However, they cause morbidity at the donor nerve site and only a limited amount of nerve can be harvested. Nerve conduits are a promising alternative to autografts and can act as guidance cues for the regenerating axons, without the need to harvest donor nerve. Separately, it has been shown that localized delivery of GDNF can enhance axon growth and motor recovery. FK506, an FDA approved small molecule, has also been shown to enhance peripheral nerve regeneration. This paper describes the design of a novel hole-based drug delivery apparatus integrated with a polytetrafluoroethylene (PTFE) nerve conduit for controlled local delivery of a protein such as GDNF or a small molecule such as FK506. The PTFE devices were tested in a diffusion chamber, and the bioactivity of the released media was evaluated by measuring neurite growth of dorsal root ganglions (DRGs) exposed to the released drugs. The drug delivering nerve guide was able to release bioactive concentrations of FK506 or GDNF. Following these tests, optimized drug releasing nerve conduits were implanted across 10 mm sciatic nerve gaps in a BL6 yellow fluorescent protein (YFP) mouse model, where they demonstrated significant improvement in muscle mass, compound muscle action potential, and axon myelination in vivo as compared with nerve conduits without the drug. The drug delivery nerve guide could release drug for extended periods of time and enhance axon growth in vitro and in vivo.     

Regulation of Gdnf expression by retinoic acid in Sertoli cells

Glial cell line‐derived neurotrophic factor (GDNF) and retinoic acid (RA) are two molecules crucial for the regulation of the spermatogonial compartment of the testis. During the cycle of the seminiferous epithelium, their relative concentration oscillates with lower GDNF levels in stages where RA levels are high. It has been recently shown that RA negatively regulates Gdnf expression but the mechanisms behind are so far unknown. Here, we show that RA directly downregulates Gdnf mRNA levels in primary murine Sertoli cells through binding of RARα to a novel DR5‐RARE on Gdnf promoter. Pharmacological inhibition and chromatin immunoprecipitation–quantitative polymerase chain reaction analysis suggested that the underlying mechanism involved histone deacetylase activity and epigenetic repression of Gdnf promoter upon RA treatment.     

胶质细胞源性神经营养因子(△N39)-R9神经营养活性和透过血脑屏障的研究

为了研究胶质细胞源性神经营养因子(GDNF)在中枢神经系统疾病中的治疗应用,运用基因突变、蛋白质融合表达和蛋白质纯化技术获得分子质量较小的GDNF(△N39)活性片段.将HIV-1 Tat蛋白转导区(protein transduction domain,PTD)的9个碱性氨基酸49RKKRRQRRR57模拟物9个精氨酸(R9)与GDNF(△N39)活性片段融合表达,获得纯度达95%以上的GDNF(△N39)-R9融合蛋白.将GDNF、GDNF(△N39)、GDNF(△N39)-R9分别加入原代培养的中脑多巴胺能神经元和转染GDNF受体GFRαl和Ret的PC12细胞中,观察它们的神经营养活性和毒性.运用脑微血管内皮细胞株B-Endo 3,观察GDNF(△N39)-R9蛋白穿越血管内皮细胞膜的功能;运用脑血管内皮细胞和Matrigel铺板模拟血脑屏障,Transwell法检测Tat-GDNF(△N39)蛋白穿越脑血管内皮细胞和外周胶质膜的能力.结果显示:GDNF(△N39)-R9蛋白具有类似GDNF的神经营养活性,促进原代培养的中脑多巴胺能神经元和稳定表达GFRα1和Ret受体的PC12-GFRα1-Ret细胞株的存活,没有显示毒性,并且能很好地穿过脑微血管内皮细胞层和模拟的血脑屏障.     

胶质细胞源性神经营养因子(ΔN39)-R9神经营养活性和透过血脑屏障的研究

为了研究胶质细胞源性神经营养因子 (GDNF) 在中枢神经系统疾病中的治疗应用,运用基因突变、蛋白质融合表达和蛋白质纯化技术获得分子质量较小的GDNF(ΔN39)活性片段. 将HIV-1 Tat 蛋白转导区 (protein transduction domain,PTD) 的9个碱性氨基酸⁴⁹RKKRRQRRR⁵⁷模拟物9个精氨酸(R9)与GDNF(ΔN39)活性片段融合表达,获得纯度达95%以上的GDNF(ΔN39)-R9融合蛋白. 将GDNF、GDNF(ΔN39)、GDNF(ΔN39)-R9分别加入原代培养的中脑多巴胺能神经元和转染GDNF受体GFRα1和Ret的PC12细胞中,观察它们的神经营养活性和毒性. 运用脑微血管内皮细胞株B-Endo 3,观察GDNF(ΔN39)-R9蛋白穿越血管内皮细胞膜的功能;运用脑血管内皮细胞和Matrigel铺板模拟血脑屏障,Transwell法检测Tat-GDNF(ΔN39)蛋白穿越脑血管内皮细胞和外周胶质膜的能力. 结果显示：GDNF(ΔN39)-R9蛋白具有类似GDNF的神经营养活性,促进原代培养的中脑多巴胺能神经元和稳定表达GFRα1和Ret受体的PC12-GFRα1-Ret细胞株的存活,没有显示毒性,并且能很好地穿过脑微血管内皮细胞层和模拟的血脑屏障.     

Differential effects of ret and TRKB on axonal branching and survival of parasympathetic neurons

Interactions between neurons and their targets of innervation influence many aspects of neural development. To examine how synaptic activity interacts with neurotrophic signaling, we determined the effects of blocking neuromuscular transmission on survival and axonal outgrowth of ciliary neurons from the embryonic chicken ciliary ganglion. Ciliary neurons undergo a period of cell loss due to programmed cell death between embryonic Days (E) 8 and 14 and they innervate the striated muscle of the iris. The nicotinic antagonist d‐tubocurarine (dTC) induces an increase in branching measured by counting neurofilament‐positive voxels (NF‐VU) in the iris between E14‐17 while reducing ciliary neuron survival. Blocking ganglionic transmission with dihyro‐β‐erythroidin and α‐methyllycacontine does not mimic dTC. At E8, many trophic factors stimulate neurite outgrowth and branching of neurons placed in cell culture; however, at E13, only GDNF stimulates branching selectively in cultured ciliary neurons. The GDNF‐induced branching at E13 could be inhibited by BDNF. Blocking ret signaling in vivo with a dominant negative (dn)ret decreases survival of ciliary and choroid neurons at E14 and prevents dTC induced increases in NF‐VU in the iris at E17. Blocking TRKB signaling with dn TRKB increases NF‐VU in the iris at E17 and decreases neuronal survival at E17, but not at E14. Thus, RET promotes survival during programmed cell death in the ciliary ganglion and contributes to promoting branching when synaptic transmission is blocked while TRKB inhibits branching and promotes maintenance of neuronal survival. These studies highlight the multifunctional nature of trophic molecule function during neuronal development. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

人脑胶质瘤GDNF基因启动子I区H3组蛋白乙酰化修饰情况

目的：明确GDNF启动子I区在人脑胶质瘤中H,赖氨酸残基9位乙酰化（H3K9Ac）情况,探讨其对于GDNF在胶质瘤中表达的影响。方法：RT-PCR检测各组中GDNFmRNA的表达;建立基于Real．timePCR分析的染色质免疫共沉淀（CHIP）方法,检测12例胶质瘤与6例正常脑组织中GDNF基因启动子I区王H3组蛋白乙酰化情况。结果：Real-timePCR验证人脑胶质瘤GDNFmRNA的表达,转录水平随级另q的增高而增高,且低级别组、高级别组与正常组之间存在显著的统计学差异（P〈0．05）。启动子I区的H,组蛋白乙酰化水平,正常组与低级别组和高级别组之间比较均有显著性差异（P〈0．05）,且低级别与高级别之间也有显著性差异。结论：在人脑胶质瘤组织中,GDNF启动子I区发生了H3组蛋白高乙酰化修饰,这种修饰很可能会影响GDNF基因的表达。     

针刺对PD胃肠功能障碍大鼠不同部位GDNF含量及胃肠生理指标的影响

摘要 目的：探讨不同针刺对帕金森（Parkinson''s Disease）胃肠功能障碍大鼠中脑黑质、胃、结肠中胶质细胞源性神经营养因子(GDNF)含量变化影响。方法：腹腔注射鱼藤酮溶液建立PD胃肠功能障碍模型并用阿普吗啡（APO）诱导实验检测。将60只已造模大鼠按随机数字表法分成模型组、假手术组、西药组、空白组、调神畅志组和常规针刺共6组,给予相应治疗2周。观察各组大鼠体重变化,检测粪便含水率、胃内容物残留率及小肠推进率,采用免疫组织化学法及ELISA法对黑质、胃、结肠GDNF含量检测。结果：体重和粪便含水率方面：西药组及两针刺组较模型组比,差异显著（P＜0.01）,西药组改善程度优于调神组（P＜0.05）,调神组改善程度优于常规组（P＜0.05）。小肠推进率和胃内容物残留率：西药组及两针刺组与模型组相比差异明显（P＜0.01）;西药组改善优于调神组（P＜0.05）,调神组优于常规组（P＜0.05）。中脑黑质、胃、结肠GDNF变化：空白组及假手术组含量高于模型组（P＜0.01）;西药组及两针刺组较模型组相比,含量升高（P＜0.01）,西药组优于调神组（P＜0.05）,调神组均优于常规组（P＜0.05）。结论：针刺治疗可提高PD胃肠功能障碍大鼠黑质、胃、结肠中GDNF含量,且调神组优于常规组,与西药组接近。调神畅志针法对PD胃肠功能障碍作用的原理可能是因为提升了结肠、脑、胃的GDNF含量来完成。     

pIRES2-GDNF-NT-3真核表达载体的构建与鉴定(英文)

目的:采用一种简便和高效的方法构建双基因共表达载体pIRES2-GDNF-NT-3。方法:人胶质细胞源性神经营养因子和神经营养素3是采用PCR的方法从人外周血单个核细胞的基因组DNA中获取,将人胶质细胞源性神经营养因子的cDNA片段插入到pIRES2-EGFP多克隆位点构建成为pIRES2-GDNF-EGFP.神经营养素3 cDNA片段通过替换EGFP的方式插入到pIRES2-GDNF-EGFP中构建成为pIRES2-GDNF-NT-3双基因共表达载体。结果:人胶质细胞源性神经营养因子和神经营养素3被克隆,通过测序和酶切鉴定的得知与基因库报道序列一致。结论:人神经生长因子和神经营养素3双基因真核表达载体成功构建,它提供了一个新的表达系统,为进一步研究双基因的功能奠定了基础。