肿瘤坏死因子α（Tumor necrosis factor alpha，TNFα）是一种重要的免疫因子，可通过调节机体昼夜节律维持稳态平衡，刺激成纤维细胞生长促进损伤和衰老组织的重塑或替换。TNFα表达紊乱已被确认为癌症、心血管疾病、自身免疫性疾病等的致病因素之一，尤其在银屑病中已作为治疗靶点被广泛研究。银屑病是一种自身免疫性疾病，因其病情反复、难以根治，严重影响患者的身体健康和生活质量。银屑病中TNFα的表达量上调，因而抑制TNFα表达被认为是最有效的治疗手段之一。但目前的治疗方法存在价格昂贵、副作用大、生物活性不稳定等缺点，亟需开发一种更为安全有效的治疗方法。通过重组病毒载体进行基因治疗是一种调节靶蛋白表达水平的新型疗法。Ⅰ 型单纯疱疹病毒（Herpes simplex virus type Ⅰ，HSV-1）作为研究最为详尽的病毒之一，由于其宿主细胞广泛且不会整合进宿主细胞基因组等优点，经过安全性病毒载体改造后，可应用于基因治疗。 本研究以探索更安全有效的银屑病基因治疗策略为出发点，针对银屑病皮损处TNFα的持续过度表达，试图利用小发夹RNA干扰技术降低TNFα 表达，以达到缓解甚至治疗银屑病的目的。为了让RNA干扰能够持续安全地在细胞中发挥作用，我们设计了一种非复制型HSV-1重组病毒载体，删除双拷贝ICP 34.5、ICP 4基因并在其潜伏转录区（LAT）插入设计好的TNFα干扰片段，通过细胞学实验检测其干扰效果。初步实验结果表明：（1）通过查阅文献及软件在线设计，得到了5条干扰序列和1条阴性对照序列；（2）通过PCR检测和测序鉴定，干扰序列均成功插入表达质粒中，分别为pnx-TNF1，pnx-TNF2，pnx-TNF3，pnx-TNF4，pnx-TNF5和pnx-MOCK；（3）将表达质粒和非复制型HSV-1病毒载体骨架DNA共转染至OG细胞中进行同源重组，发生同源重组并通过筛选的重组病毒载体有4个，分别为WD-TNF3，WD-TNF4，WD-TNF5和WD-MOCK，其余两个未成功；（4）利用结晶紫染色法对扩大纯化后病毒载体进行活性定量检测，4个病毒载体均呈现很高的滴度；（5）利用CCK8法进行转染后的细胞活力测定，确定MOI = 1，24 h为最佳转染剂量和时间，对细胞活力无显著影响；（6）利用LPS诱导细胞炎症模型检测重组病毒载体对TNFα的干扰效果，WD-TNF3为人源片段的重组病毒载体，但人角质生成细胞HaCaT的LPS炎症诱导模型失败，因此未进行WD-TNF3干扰效果的检测。WD-TNF4，WD-TNF5为鼠源片段的重组病毒载体，采用100 ng/mL LPS诱导的RAW 264.7细胞炎症模型进行实验，在MOI = 1时，基因水平检测均未呈现出很好的干扰效果。将MOI增大至最大剂量5时，WD-TNF4依然没有很好的干扰效果，WD-TNF5处理后的TNFα基因表达和 Ⅱ 型TNFα前体膜蛋白显著下降，可溶性TNFα蛋白表达增多。 本研究进行了携带干扰片段的非复制型HSV-1载体的构建，完善了病毒载体的构建过程，并成功诱导出RAW 264.7细胞炎症模型，为后续实验打下了实践基础。

Tumor necrosis factor alpha (TNFα) is an important immune factor that maintains homeostasis by regulating the body's circadian rhythm, stimulating fibroblast growth and promoting the remodeling or replacement of damaged and aged tissues. The disorder expression of TNFα has been identified as one of the pathogenic factors of cancer, cardiovascular disease, autoimmune disease, etc., especially in psoriasis, which has been widely studied as a therapeutic target. Psoriasis is an autoimmune disease, because its condition repeated, difficult to cure, seriously affect the patient's health and quality of life. The expression of TNFα was up-regulated in psoriasis, and inhibition of TNF was considered as one of the most effective treatments. However, the current treatment is expensive, side effects, biological instability and other shortcomings, it is urgent to develop a more safe and effective treatment. Gene therapy by recombinant virus vector is a novel therapy to regulate the expression level of target protein. Ⅰ Herpes simplex virus (Herpes simplex virus get type Ⅰ , HSV-1) as one of the most detailed virus, due to its host cells are widely and do not integrate into the host cell genome, after security virus vector transformation can be applied to gene therapy. This study aimed to explore a safer and more effective gene therapy strategy for psoriasis, aiming at the constant overexpression of TNFα in psoriasis lesions, and attempted to use small hairpin RNA interference technology to decrease the expression of the TNFα, so as to alleviate and even treat psoriasis. In order to allow RNA interference to continue to function safely in cells, we designed a non-replicative HSV-1 recombinant virus vector, in which double-copy ICP 34.5 and ICP 4 genes were deleted and TNFα interfering fragments were inserted into the LAT region, and their interfering effects were detected by cytological experiments. Preliminary experimental results showed that :(1) Five interference sequences and one negative control sequence were obtained through literature review and online software design. (2) pnx-TNF1, pnx-TNF2, pnx-TNF3, pnx-TNF4, pnx-TNF5 and pnx-mock were successfully inserted into the expression plasmids through PCR detection and sequencing identification. (3) The expressed plasmids and non-replicating HSV-1 virus vector skeleton DNA were co-transfected into OG cells for homologous recombination. Four recombinant virus vectors were selected for homologous recombination, namely WD-TNF3, WD-TNF4, WD-TNF5 and WD-MOCK. The other two were not successful. (4) Crystal violet staining method was used for quantitative detection of the activity of virus vectors after expanded purification. (5) CCK8 method was used to determine the cell viability after transfection, and MOI = 1, 24 h was determined as the optimal transfection dose and time, with no significant effect on cell viability. WD-TNF3 was the recombinant viral vector of human fragments. However, the LPS inflammatory induction model of human keratinocytes HaCaT failed. Therefore, WD-TNF3 interference effect was not detected. WD-TNF4 and WD-TNF5 were recombinant viral vectors of mouse fragments, and 100 ng/mL LPS-induced RAW 264.7 cell inflammatory model was used for experiments. At MOI = 1, gene level detection showed no good interference effect. Increasing MOI to maximum dose 5, WD-TNF4 is still not very good interference effect, WD-TNF5 treatment TNF alpha gene expression and Ⅱ TNF alpha precursor membrane protein decreased significantly, soluble TNF alpha protein was increased. In this study, the construction of a non-replicative HSV-1 vector with interfering fragments was carried out . Improved the construction process of the virus vector, and successfully induced the RAW 264.7 cell inflammation model, laying a practical foundation for the subsequent experiments.