重组溶瘤单纯疱疹病毒oHSV2治疗结直肠癌的实验研究

Preclinical evaluation of recombinant herpes simplex virus oHSV2 in colorectal cancer

目的:评估重组溶瘤单纯疱疹病毒oHSV2在CT26结肠癌荷瘤小鼠中的抗肿瘤效果,初步探讨其治疗机制。方法:构建CT26细胞荷瘤小鼠肿瘤模型。1)小鼠瘤内注射oHSV2,ELISA法测定血清中粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)浓度;2)荷瘤小鼠分为oHSV2组、5-氟尿嘧啶(5-FU)组(阳性对照组)、磷酸盐缓冲液(PBS)组(阴性对照组)。给药后,记录小鼠体质量、肿瘤体积、生存期及生活状态等变化,评估病毒抗肿瘤效果;3)流式细胞术定量检测肿瘤引流淋巴结(tumor-draining lymphnode,TDLN)内树突状细胞(dendritic cell,DC)及瘤体内CD4+T与CD8+T的比例。结果:1)瘤内注射oHSV2后,血清中的GM-CSF浓度不断升高。首次给药后第8天出现高峰(3150±327.1)pg/m L,后缓慢下降;2)相比PBS组,oHSV2和5-FU组均表现出显著的抗肿瘤效果,小鼠生存期显著延长(50 d vs.36 d,P<0.01;51 d vs.36 d,P<0.01),但oHSV2治疗未引起小鼠体质量下降。治疗起始第28天,5-FU组平均体质量较PBS组有显著性差异(16.61 g vs.22.07 g,P<0.01),oHSV2组与PBS组差异无统计学意义(P>0.05),且小鼠病毒注射区皮肤无坏死、溃疡;3)流式分析结果显示相比PBS组,oHSV2治疗组的DC(6.49%vs.3.73%,P<0.01),CD4+T(15%vs.8.57%,P<0.01)与CD8+T(8.19%vs.5.15%,P<0.01)比例升高。而5-FU组各细胞比例较PBS组明显降低(P<0.05)。结论:瘤内注射oHSV2有效抑制结直肠癌细胞生长,病毒治疗与化疗药物相比不伴有明显的毒副反应。病毒在荷瘤小鼠体内复制产生具有生物活性的GM-CSF,可增强抗肿瘤免疫。

Objective： To investigate therapeutic efficacy and mechanisms of action of oncolytic agent derived from herpes simplex virus type 2 （oHSV2） in a xenograft mouse model bearing CT26 colorectal cancer. Methods： BALB/c mice were subcutaneously inoculated with CT26 cells to establish a xenograft mouse model of colorectal cancer. 2） After intratumoral administration of oHSV2, enzyme-linked im- munosorbent assay was used to determine granulocyte-macrophage colony-stimulating factor （GM-CSF} expression levels in the blood. 2） Model mice were divided into three groups： PBS group （negative control）, oHSV2 group, and 5-fluorouracil （5-FU） group （positive control）. After drug administration, drug effectiveness was evaluated on the basis of weight, tumor volume, general state, and survival time. 3） Cells from the draining lymph nodes （TDLN） and tumor were surgically removed and used to quantify mature dendritic cells （DCs） and T lymphocytes by flow cytometry. Result： 2） In the CT26 xenograft model, level of GM-CSF continuously elevated. At day 8, peak value was attained in the blood at concentration of 3150±327.1 pg/mL. Then, GM-CSF expression gradually reduced as time progressed. 2） In in vivo study, both oHSV2 and 5-FU exerted antitumor effects relative to PBS group （50 days vs. 36 days, P〈0.01; 51 days vs. 36 days, P〈0.01）, and oHSV2 proved to be less toxic and safer. At day 28, the 5-FU group presented highly significant difference in mouse body weight compared with that of PBS group （26.61 g vs. 22.07 g, P〈0.01）. However, oHSV2 group did not show statistically significant change （all P〉0.05）. Skin of virus injection region did not present necrosis and ulceration. 3） In the TDLN, the frequency of DC was increased when treated with oHSV2 compared with the control group （6.49% vs. 3.73%, P〈0.01）. Similarly, the percentage of CD4^＋ and CD8^＋T-cells from the oHSV2-treated group was signifcantly higher than mock-treated tumors （15% vs. 8.57%, P〈0.01; 8.19% vs. 5.15%, P〈0.01）. However, number of cells in the 5-FU group were significantly reduced with respect to that of the negative group （all P〈0.01）. Conclusion： oHSV2 exerted potent antitumor effects in a murine colorectal cancer model. Compared with 5-FU, oHSV2 treatment caused fewer side effects. Such antitumor effect may be induced by stimulation of immune activity by GM-CSF production.