Electroporation-mediated gene delivery of cleavage-resistant pro–nerve growth factor causes retinal neuro- and vascular degeneration
Electroporation-mediated gene delivery of cleavage-resistant pro–nerve growth factor causes retinal neuro- and vascular degeneration
复制标题
电穿孔介导的抗裂解前神经生长因子基因传递导致视网膜神经和血管变性
DOI：
10.9734/bpi/mono/978-93-5547-253-3/ch8
复制
发表时间：
2012
期刊：
影响因子：
2.2
通讯作者：
A. El
中科院分区：
文献类型：
--
作者：
S. Matragoon;Mohammed M H Al;B. Mysona;M. Abdelsaid;B. Pillai;K. Neet;S. Fagan;A. El
目的神经营养因子（Neurotrophins，NGF）是神经胶质细胞分泌的神经生长因子前体（pro-form，proNGF）。在视网膜神经退行性疾病中，proNGF的增加已被充分记录。由于proNGF的系统性过表达表现出胚胎致死性，我们的目的是建立一个模型，特异性和稳定的过表达的抗切割突变体的proNGF（proNGF 123）质粒在视网膜中使用电穿孔。方法将pGFP或pGFP-proNGF 123质粒分别注入SD大鼠玻璃体内，然后在不同条件下进行电穿孔。视网膜细胞死亡和神经节细胞计数通过TUNEL和抗Brn 3免疫染色进行评估。蛋白质印迹法检测proNGF、NGF及其受体的表达。通过牛血清白蛋白-荧光素外渗来评估视网膜血管通透性。通过高碘酸-希夫和苏木精染色评估无细胞毛细血管的发育。结果pGFP-proNGF 123基因转染成功，proNGF在视网膜组织中表达，Western blot和免疫组化结果显示proNGF在视网膜组织中表达。proNGF的过表达降低了NGF的表达，同时诱导了神经营养因子受体的表达，包括p75 NTR和酪氨酸受体激酶A，但不包括分拣蛋白。proNGF过表达导致大鼠视网膜神经节细胞计数减少约50%，TUNEL阳性细胞增加5倍。此外，proNGF的过度表达诱导血-视网膜屏障的破坏，1周后牛血清白蛋白-荧光素外渗增加两倍，4周后诱导无细胞毛细血管的发育。结论电穿孔法可以成功地在大鼠视网膜局部整合和表达具有生物活性的抗切割proNGF。抗切割proNGF的过表达可以成为研究proNGF引起视网膜神经变性和血管损伤的特定分子机制的有用工具。
Purpose Neurotrophins, including nerve growth factor (NGF), are secreted by glia as a pro-form (proNGF) that is normally cleaved into the mature ligand. Increases of proNGF has been well documented in retinal neurodegenerative diseases. Since systemic overexpression of proNGF exhibits embryonic lethality, we aimed to establish a model that specifically and stably overexpresses a cleavage-resistant mutant of proNGF (proNGF123) plasmid in the retina using electroporation. Methods Male Sprague-Dawley rats were injected intravitreally with pGFP or pGFP-proNGF123 plasmids, then electroporated with various settings for optimization. Retinal cell death and ganglion cell count were assessed by TUNEL and immunostaining with anti-Brn3. Expression of proNGF, NGF, and their receptors was examined by western blot. Retinal vascular permeability was assessed by extravasation of bovine serum albumin–fluorescein. Development of acellular capillaries was assessed by periodic acid-Schiff and hematoxylin staining. Results Successful pGFP-proNGF123 gene delivery and expression of proNGF was demonstrated by western blot and extensive proNGF immunostaining in retina sections. Overexpression of proNGF reduced NGF expression while inducing the expression of neurotrophin receptors, including p75NTR and tyrosine receptor kinase A, but not sortilin. Overexpression of proNGF resulted in ~50% reduction in ganglion cell count and fivefold increase in TUNEL-positive cells in rat retina. In addition, overexpression of proNGF induced breakdown of the blood-retina barrier evident by twofold increase in extravasation of bovine serum albumin–fluorescein after 1 week and induced the development of acellular capillaries after 4 weeks. Conclusions Electroporation can successfully incorporate and express biologically active cleavage-resistant proNGF locally in rat retinas. Overexpression of cleavage-resistant proNGF can be a useful tool to investigate specific molecular mechanisms by which proNGF causes neurodegeneration and vascular injury in the retina.
影响因子：
4.4
作者：
Zheng, Ling;Gong, Bendi;Kern, Timothy S.
通讯作者：
Kern, Timothy S.
影响因子：
6
作者：
El-Remessy, AB;Behzadian, MA;Caldwell, RB
通讯作者：
Caldwell, RB