表达单纯疱疹病毒受体HVEM的黑色素瘤B16-ova-HVEM细胞系的构建及成瘤后oHSV2治疗效果初探

The construction of a herpes simplex virus-permissive mouse melanoma cell line B16-ova-HVEM and the therapeutic efficacy of oHSV2 for B16-ova-HVEM

目的构建可被单纯疱疹病毒感染的新型细胞系B16-ova-HVEM,体外验证目的基因的表达并初步探究其成瘤后,应用Ⅱ型溶瘤单纯疱疹病毒(oHSV2)的治疗效果。方法构建表达单纯疱疹病毒受体(HVEM)的质粒载体,脂质体转染B16-ova细胞,嘌呤霉素筛选表达HVEM的阳性克隆。体外验证后进行体内检测,B16-ova-HVEM荷瘤C57BL/6小鼠,成瘤后分为两组,每组5只小鼠,给予oHSV2治疗为oHSV2治疗组,荷瘤未治疗的小鼠为对照组。治疗后测量肿瘤大小,观察小鼠生存期;流式细胞术检测两组小鼠外周血中CD4^+、CD8^+T细胞和髓系来源的抑制性细胞(MDSC)比例变化。结果质粒DNA测序和酶切鉴定结果均显示成功构建含HVEM的质粒;荧光显微镜下可观察到筛选后细胞发绿色荧光;流式细胞术检测筛选后细胞GFP阳性率为98.3%;RT-PCR验证结果显示筛选后细胞含有目的基因HVEM;oHSV2在感染复数(MOI)为0.1、0.5和1的情况下均能明显感染筛选出的细胞;动物实验显示,oHSV2体内治疗后抑瘤效果显著(P﹤0.001);流式检测结果显示oHSV2治疗后小鼠外周血中CD4^+、CD8^+T细胞比例明显高于对照组(P﹤0.01),而MDSC比例明显低于对照组(P﹤0.01)。结论成功构建B16-ova-HVEM细胞系,其既可被OT-1小鼠T细胞特异性识别又可被单纯疱疹病毒感染,为肿瘤特异性免疫治疗和溶瘤病毒联合治疗的研究奠定基础。

Objective The aim of this study was to construct a oncolytic herpes simplex virus permissive mouse melanoma cell line B16-ova-HVEM. Then the expression of HVEM gene and the efficacy of oHSV2 therapy were deter-mined. Method The plasmid vector expressing HVEM was constructed and transfected into B16-ova cells via lipo-some. Positive cells expressing HVEM were selected by puromycin. The confirmed B16-ova-HVEM cells were subcuta-neously inoculated into the C57/BL mouse. Tumor-bearing mice （n=5） were treated with oHSV2 after tumor formation and compared with untreated group （n=5）. The tumor volume was measured and mice survival was recorded;Flow cy-tometry was applied to detect the CD4＋, CD8＋T cells and MDSC proportion in all mice’s peripheral blood. Result We have successfully constructed the plasmid expressing HVEM, which was verified by plasmid DNA sequencing and en-zyme digestion. Green fluorescent can be viewed in the selected cells using fluorescence microscope. GFP-positive rate of the selected cells was 98.3%through flow cytometry. RT-PCR showed that most of the selected cells were obviously infected by oHSV2 when MOI were 0.1, 0.5 and 1. oHSV2 could significantly inhibit the growth of B16-ova-HVEM in vivo （P〈0.001）. The percentage of CD4＋and CD8＋T cells in the peripheral blood of the mice treated with oHSV2 were significantly higher than that of control group while the percentage of MDSC was lower （P〈0.01）. Conclusion We construct the new B16-ova-HVEM cell line, which can not only be specifically killed by the T cell from OT-1 mice but also can be infected by herpes simplex virus. This new cell line lays the foundation of the combination of tumor specific immune therapy and oncolytic virus therapy.