南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (10): 1448-1455.doi: 10.12122/j.issn.1673-4254.2021.10.02

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潜伏表达癌调蛋白的HSV-1减毒载体能有效治疗大鼠机械性视神经损伤

杨 明，高瀛政，李 萌，曹 霞，黄新伟   

  1. 昆明医科大学第一附属医院眼科，云南 昆明 650032；昆明医科大学第二附属医院，云南 昆明 650033
  • 出版日期:2021-10-20 发布日期:2021-11-11

Attenuated Herpes simplex virus 1 vector expressing oncomodulin effectively allieviates mechanical optic nerve injury in rats

YANG Ming, GAO Yingzheng, LI Meng, CAO Xia, HUANG Xinwei   

  1. Department of Ophthalmology, First Affiliated Hospital of Kunming Medical University, Kunming 650032, China; Second Affiliated Hospital of Kunming Medical University, Kunming 650033, China
  • Online:2021-10-20 Published:2021-11-11

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摘要： 目的 评价潜伏表达癌调蛋白OCM的HSV-1减毒型载体（1716-OCM）在大鼠机械性视神经损伤中的治疗作用及安全性。方法 体外细胞感染模型中检测重组病毒增殖特性及OCM表达。大鼠按随机数字表法分为3组（对照组、1716-OCM注射组和野生型病毒角膜感染组），每时间点3只/组，于感染后7、14、30、60 d免疫荧光法检测OCM基因及病毒蛋白gB在大鼠视网膜和下丘脑中的表达。大鼠视神经损伤模型中，大鼠按随机数表法分为4组（假手术组，PBS治疗对照组，1716-OCM感染组及1716-OCM感染加cAMP增敏组），5只/组。治疗45 d后，视觉诱发电位（FVEP）检测视觉电生理功能。免疫荧光法检测视网膜RGCs数量及神经髓鞘蛋白表达。结果 重组减毒型1716-OCM在体外细胞感染中病毒增殖远低于野生型病毒，其能够介导OCM有效表达。重组病毒能够介导OCM在大鼠眼部RGC层及脉络膜层表达。相比于野生型病毒，1716-OCM未引发显著的眼部组织结构破坏。在视神经损伤模型中，相比于未治疗组，1716-OCM联合cAMP治疗能够显著促进视网膜RGC存活（P=0.007）并抑制视神经脱髓鞘（P=0.03）。FVEP分析显示1716-OCM联合cAMP显著促进大鼠ΔN1-P1峰振幅恢复（P<0.001）。结论 减毒型重组1716-OCM能够介导OCM在大鼠视网膜表达，玻璃体腔注射1716-OCM重组病毒和cAMP能够有效治疗大鼠机械性视神经损伤。

关键词: I型单纯疱疹病毒, 潜伏表达, 视神经损伤, 癌调蛋白

Abstract: Objective To evaluate the efficacy and safety of attenuated Herpes simplex virus 1 (HSV-1) vector expressing oncomodulin (OCM) for treatment of mechanical optic nerve injury in rats. Methods The proliferation characteristics and OCM expression of the recombinant HSV-1 vector (1716-OCM) was assessed in cultured Vero cells. Twelve-week-old SD rats were randomly divided into control group, 1716-OCM injection group and wild-type virus corneal infection group, and at 7, 14, 30 and 60 days post-infection (3 rats in each group at each time point), the expressions of OCM and HSV-1 structural protein gB in the retina and the hypothalamus of the rats were detected using immunofluorescence assay. Another 20 rats were randomized into sham operation group, PBS treatment group, 1716-OCM infection group and 1716-OCM infection with cAMP sensitization group (n=5), and in the latter 3 groups, rat models of optic nerve injury models were established followed by intravitreal injection of PBS, 1716-OCM or cAMP as indicated. At 45 days after the treatments, the rats were examined for visual electrophysiological function using FVEP method, and the number of retinal ganglion cells (RGCs) and the expression of myelin basic protein in the optic nerve were detected using immunofluorescence assay. Results The recombinant 1716-OCM vector was capable of mediating effective expression of OCM in Vero cells in vitro, but its proliferation rate was much lower than that of the wild-type virus. In SD rats, the recombinant virus could mediate the expression of OCM in the RGC layer and choroid layer of the eyes without inducing significant structural damage of the eyes as compared with the wild-type virus. In rat models of optic nerve injury, 1716-OCM combined with cAMP significantly promoted the survival of retinal RGCs (P= 0.007) and inhibited demyelination of the optic nerve (P=0.03) as compared with the mock treatment. FVEP analysis showed that 1716-OCM combined with cAMP significantly promoted the recovery of the peak amplitude of ΔN1-P1 in the rats (P< 0.0001). Conclusion Attenuated recombinant 1716-OCM vector can mediate OCM expression in the retina of rats, and in rat models of mechanical optic nerve injury, intravitreal injection of 1716-OCM combined with cAMP can effectively alleviate optic nerve injuries.

Key words: Herpes simplex virus 1, latent expression, mechanical optic nerve injury, oncomodulin