嵌合IL-21的重组溶瘤流感病毒构建及其在肝癌中的溶瘤机制

曹蕊; 曾桂能; 田昱莹; 李聪; 肖瑞雪; 徐永茹; 杨鹏辉; 康毅敏

doi:10.16016/j.2097-0927.202407051

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嵌合IL-21的重组溶瘤流感病毒构建及其在肝癌中的溶瘤机制

基础医学 | 更新时间：2026-01-09

- 嵌合IL-21的重组溶瘤流感病毒构建及其在肝癌中的溶瘤机制
- Construction of chimeric IL-21 recombinant oncolytic influenza virus and mechanisms for its antitumor efficacy against hepatocellular carcinoma
- 陆军军医大学学报 2024年46卷第24期页码：2736-2744
- 作者机构：
  
  1.010110 呼和浩特，内蒙古医科大学基础医学院
  2.100853 北京，中国人民解放军总医院第一医学中心肝胆胰外科医学部研究所
- 作者简介：
  
  [康毅敏，E-mail: kangym@immu.edu.cnKANG Yimin, E-mail: kangym@immu.edu.cn
- 基金信息：
- DOI：10.16016/j.2097-0927.202407051
  中图分类号： R392.2;R730.51;R735.7
- 收稿：2024-07-12，
  
  修回：2024-09-12，
  
  纸质出版：2024-12-30
- 稿件说明：
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曹蕊, 曾桂能, 田昱莹, 等. 嵌合IL-21的重组溶瘤流感病毒构建及其在肝癌中的溶瘤机制[J]. 陆军军医大学学报, 2024,46(24):2736-2744.

Rui CAO, Guineng ZENG, Yuying TIAN, et al. Construction of chimeric IL-21 recombinant oncolytic influenza virus and mechanisms for its antitumor efficacy against hepatocellular carcinoma[J]. Journal of Army Medical University, 2024, 46(24): 2736-2744.
曹蕊, 曾桂能, 田昱莹, 等. 嵌合IL-21的重组溶瘤流感病毒构建及其在肝癌中的溶瘤机制[J]. 陆军军医大学学报, 2024,46(24):2736-2744. DOI： 10.16016/j.2097-0927.202407051.

Rui CAO, Guineng ZENG, Yuying TIAN, et al. Construction of chimeric IL-21 recombinant oncolytic influenza virus and mechanisms for its antitumor efficacy against hepatocellular carcinoma[J]. Journal of Army Medical University, 2024, 46(24): 2736-2744. DOI： 10.16016/j.2097-0927.202407051.

摘要

目的

拯救嵌合白细胞介素-21(interleukin-21

IL-21)的重组溶瘤流感病毒

评价其对肝细胞癌的抑制效果及安全性

并探索其联合程序性死亡受体1(programmed cell death-protein 1

PD-1)抗体增强抗肿瘤作用的机制。

方法

将IL-21基因片段插入流感病毒PR8的非结构蛋白(nonstructural protein

NS)序列

利用反向遗传学(reverse genetics

RG)技术

拯救重组溶瘤流感病毒rOV-IL-21-NS;采用半数组织培养感染剂量(50% tissue culture infectious dose

TCID50)和血凝实验测定病毒滴度和毒力;采用反转录定量聚合酶链反应(reverse transcription quantitative polymerase chain reaction

RT-qPCR)、凝胶电泳和测序分析验证外源基因是否成功插入NS序列;通过透射电镜观察病毒形态特征及大小

采用CCK-8检测其对肝癌细胞活力的影响;建立C57BL/6小鼠肝细胞癌皮下荷瘤模型

将45只8周龄雌性C57BL/6小鼠(体质量16~20 g)按简单随机分组法分为5组(每组9只):PBS组、PR8组、PD-1抗体组、rOV-IL-21-NS组和rOV-IL-21-NS联合PD-1抗体治疗组

以评价单药和联合疗法抗肿瘤效果;通过流式细胞术评价单药和联合疗法对肿瘤免疫微环境的调控作用。

结果

利用RG技术成功拯救重组溶瘤流感病毒rOV-IL-21-NS

经测序验证了IL-21成功插入目的序列

其可稳定传代

第6代病毒血凝效价达2

病毒滴度达10

TCID50/mL。rOV-IL-21-NS选择性降低肝癌细胞活力而对正常肝细胞无明显影响。相对于PBS组

rOV-IL-21-NS联合PD-1抗体显著抑制小鼠肿瘤的生长(

0.001)

增加脾脏组织中CD4

CD69

T及CD8

CD69

T细胞的比例(

0.05)。

结论

嵌合IL-21的重组溶瘤流感病毒rOV-IL-21-NS可有效、安全地靶向杀伤肝癌细胞

协同PD-1抗体进一步增强T细胞活化

改善免疫微环境。

Abstract

Objective

To rescue a recombinant oncolytic influenza virus chimeric with IL-21 and evaluate its inhibitory effects and safety against hepatocellular carcinoma (HCC) both

in vitro

and

in vivo

and to explore the mechanism by which this virus

enhances antitumor effects when combined with anti-programmed cell death protein 1(anti-PD-1) antibody.

Methods

The IL-21 gene fragment was inserted into the nonstructural protein (NS) sequence of the influenza virus PR8 using reverse genetics (RG) technology to rescue the recombinant oncolytic influenza virus rOV-IL-21-NS.The virus titer and virulence were determined using the 50% tissue culture infectious dose (TCID50) and hemagglutination assays.The successful insertion of the exogenous gene into the NS sequence was verified using RT-qPCR

gel electrophoresis

and sequencing analysis.Viral morphology and size were observed with transmission electron microscopy.The impact of the virus on the viability of hepatocellular carcinoma cells was assessed with CCK-8 assay.A subcutaneous tumor model of HCC was established in 45 female C57BL/6 mice (8 weeks old

weighing 16~20 g)

and then the mice were randomly assigned into PBS

PR8

anti-PD-1

rOV-IL-21-NS

and the rOV-IL-21-NS+anti-PD-1 treatment groups

with 9 mice in each group

to evaluate the anti-tumor effects of monotherapy versus combination therapy.Flow cytometry was conducted to assess the regulatory effects of monotherapy and combination therapy on the tumor immune microenvironment.

Results

RG technology successfully rescued the recombinant oncolytic influenza virus rOV-IL-21-NS.Sequencing confirmed the successful insertion of IL-21 into the target sequence

and the obtained virus could be stably propagated

with its sixth passage reaching a hemagglutination titer of 2

and a viral titer of 6 Log

(TCID50/mL).Oncolytic virus rOV-IL-21-NS

at a multiplicity of infection (MOI) of 3

selectively reduced the viability of HCC cells without significantly affecting normal liver cells.Compared to the control group

the combination of rOV-IL-21-NS and anti-PD-1 antibodies significantly inhibited tumor growth (

0.001) and increased the proportions of CD4

T and CD8

T cells in the spleen tissue of the mouse model of subcutaneous HCC tumor (

0.001).

Conclusion

The recombinant oncolytic influenza virus rOV-IL-21-NS chimeric with IL-21 can effectively and safely exert targeted killing to HCC cells

enhance T cell activation by synergizing with anti-PD-1 antibodies

and improve the immune microenvironment.

关键词

Keywords

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