溶瘤Ⅱ型单纯疱疹病毒在肿瘤中的复制及表达

Replication and expression of oncolytic herpes simplex virus type Ⅱ in tumors

摘要:

本研究旨在检测BALB/c小鼠肿瘤注射部位溶瘤Ⅱ型单纯疱疹病毒(oncolytic herpes simplex virus type 2, oHSV2)的持续时间和复制水平。同时检测血清中人粒细胞-巨噬细胞集落刺激因子(human granulocyte macrophage colony-stimulating factor, hGM-CSF)的表达量和HSV-2抗体水平。小鼠经瘤内注射高、低两个剂量oHSV2-Fluc[萤火虫荧光素酶(firefly luciferase, Fluc)]，检测其荧光表达变化及持续时间。采用实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qPCR)检测肿瘤组织中oHSV2基因拷贝数。酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)检测血清中hGM-CSF表达量以及HSV-2抗体水平。结果表明高剂量组肿瘤体积显著小于对照组(P<0.01)。瘤内注射oHSV2-Fluc表明病毒携带的Fluc可以在肿瘤中持续表达，于第11天仍能检测到荧光，直至第18天低于检测线。通过提取肿瘤组织RNA进行qPCR，表明第9天高、低剂量组肿瘤组织均存在oHSV2的mRNA。对血清进行ELISA检测，相较于对照组，高、低剂量组hGM-CSF及HSV2抗体水平显著提高(P<0.05)。这些发现表明，oHSV2能够在瘤内正常复制，同时所携带的外源因子可持续表达，对肿瘤产生杀伤作用。瘤内注射oHSV2后，小鼠血清中存在较高水平的hGM-CSF和HSV-2抗体。

关键词:溶瘤病毒;持续时间;肿瘤;人粒细胞-巨噬细胞集落刺激因子

Abstract:

This study aimed to measure the duration and replication level of oncolytic herpes simplex virus type 2 (oHSV2) at the tumor injection site in BALB/c mice. Additionally, the expression level of human granulocyte macrophage colony-stimulating factor (hGM-CSF) and HSV-2 antibody in the serum was also measured. High and low doses of oHSV2-Fluc (firefly luciferin, Fluc) were injected into the mice’s tumors to track the change and duration of fluorescence expression. The copy number of oHSV2 gene in tumor tissues was determined using quantitative real-time polymerase chain reaction (qPCR). Enzyme linked immunosorbent assay (ELISA) was used to detect the expression of hGM-CSF and HSV-2 antibody in the serum. The tumor volume in the high-dose group was significantly lower than that in the control group (P<0.01). Intratumor injection of oHSV2-Fluc showed that the carried Fluc could continue to express in the tumor, with fluorescence still detectable at day 11 and declining to undetectable level by day 18. The mRNA expression of oHSV2 was detected in tumor tissues of both high and low dose groups on day 9 using qPCR. ELISA results showed that the levels of HSV2 antibody and hGM-CSF in both high and low dose groups were significantly increased compared to the control group (P<0.05) after collecting orbital blood. These findings suggest that oHSV2 can replicate in the tumor and sustainably express exogenous factors, thus effectively targeting and killing the tumor. Furthermore, intratumoral injection of oHSV2 resulted in higher levels of hGM-CSF and HSV-2 antibodies found in the mice’s serum.

Key words:oncolytic virus;duration;tumor;human granulocyte macrophage colony-stimulating factor