Establishment of a novel therapeutic vector targeting the trigeminal ganglion in rats.
Establishment of a novel therapeutic vector targeting the trigeminal ganglion in rats.
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大鼠三叉神经节新型治疗载体的建立
DOI：
10.2147/dddt.s96730
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Zheng X
中科院分区：
文献类型：
--
作者：
Xu K;Pan SY;Song JX;Liu XN;An N;Zheng X
背景在单纯疱疹病毒性角膜炎的发病机制中，单纯疱疹病毒1型(HSV-1)感染开始于角膜上皮细胞，然后通过感觉神经末梢传播到三叉神经节(TG)，在那里它仍然是潜伏的病毒。现有的抗HSV治疗并不能完全抑制活动性HSV-1感染的复发。本研究的目的是构建一种能有效靶向TG的新型复制缺陷型单纯疱疹病毒1型(rdHSV-γ)载体。方法将重组单纯疱疹病毒1型病毒插入携带干扰素γ的穿梭载体中，构建重组单纯疱疹病毒干扰素γ载体。用SH-SY5Y转基因神经母细胞瘤细胞体外半数细胞毒试验和Kaplan-Meier生存评估及感染率评价其安全性。用重组单纯疱疹病毒干扰素γ免疫Wistar大鼠，评价其对TG的靶向性。实时定量聚合酶链式反应和免疫印迹法检测干扰素γ基因和蛋白的表达及重组单纯疱疹病毒干扰素γ定位。结果成功构建了重组单纯疱疹病毒干扰素γ载体，具有较高的体外安全性和整体存活率，角膜感染率与生理盐水对照组相近(P&gT0.05)。实时定量聚合酶链式反应和免疫组织化学检测证实，免疫后第14天和第21天有干扰素γ的表达和有效的甘油三酯靶向，且随着免疫后时间的延长而增加。此外，干扰素γ在TG组织中有充分表达。结论重组单纯疱疹病毒干扰素γ可作为一种有效的基因转移载体，将治疗基因携带到甘油三酯中并触发其表达。
Background In the pathogenesis of herpes simplex keratitis, herpes simplex virus type 1 (HSV-1) infection begins in corneal epithelium cells and then progresses through the sensory nerve endings and finally travels up forward to the trigeminal ganglion (TG), where it remains as latent virus. The available anti-HSV therapies do not completely suppress the recurrence of active HSV-1 infection. The aim of this study was to establish a novel replication-defective (rd) HSV-1 (rdHSV) vector (rdHSV-interferon gamma [IFNγ]) that could effectively target the TG. Methods Recombinant HSV-1 virus was inserted into a shuttle plasmid carrying IFNγ to establish the rdHSV-IFNγ vector. Safety was evaluated in vitro by 50% cellular cytotoxicity in transfected SH-SY5Y neuroblastoma cells and in vivo by Kaplan–Meier survival estimate and infection rate. Wistar rats were immunized with rdHSV-IFNγ to evaluate the TG targeting efficiency. Real-time polymerase chain reaction and Western blot assays were used to evaluate IFNγ mRNA and protein expression and rdHSV-IFNγ localization. Results The rdHSV-IFNγ vector was successfully constructed and showed high in vitro safety and overall survival and a corneal infection rate similar to that of control rats immunized with saline (control group; P>0.05). Real-time polymerase chain reaction and immunohistochemistry assays confirmed IFNγ expression and effective TG targeting on days 14 and 21, which increased with postimmunization time. Moreover, IFNγ was expressed sufficiently in the TG tissues. Conclusion The rdHSV-IFNγ can act as an effective gene transporting vector that carries the therapeutic genes to the TG and triggers its expression.