表达IL-21的重组溶瘤流感病毒在胰腺癌中的抗肿瘤及免疫激活作用研究

doi:10.3877/cma.j.issn.2095-1221.2026.03.001

目的

探讨表达白细胞介素-21 （IL-21）的重组溶瘤流感病毒rOV-IL-21在胰腺癌中的治疗潜力。

方法

通过血凝实验及CCK-8评估rOV-IL-21在胰腺癌细胞中复制、IL-21表达及细胞杀伤效应。建立C57BL/6小鼠胰腺癌皮下荷瘤模型，监测肿瘤生长，通过流式细胞术评估病毒在肿瘤内免疫激活作用，并对小鼠体重、血清ALT、AST水平及主要脏器的组织学变化监测进行体内安全性评估。多组间比较采用单因素方差分析，组间两两比较采用Tukey多重比较检验；对于涉及时间和处理剂量的比较时，采用两因素重复测量方差分析。

结果

rOV-IL-21在胰腺癌细胞中能够稳定复制并有效表达IL-21，且具有明显的剂量依赖性杀伤作用。在小鼠胰腺癌细胞KPC同源移植小鼠皮下瘤模型中，rOV-IL-21抑制肿瘤生长（P < 0.001），延长小鼠生存（P = 0.001 8）。与对照相比，rOV-IL-21增强CD8⁺ T细胞的杀伤性功能，增加GzmB⁺CD8⁺ T细胞比例[（19.23 ± 1.46）%比（7.53 ± 2.28）%]，减少耗竭性CD8⁺ T细胞比例[（3.47 ± 0.52）%比（14.54 ± 5.91）%]，差异有统计学意义（P 均 < 0.05）；rOV-IL-21组小鼠血清谷丙转氨酶及谷草转氨酶活性水平差异无统计学意义（P > 0.05）。

结论

rOV-IL-21在胰腺癌模型中表现出良好的安全性和显著的抗肿瘤效果，与增强肿瘤内CD8⁺ T细胞的细胞毒功能有关，为胰腺癌免疫治疗提供潜在溶瘤病毒策略。

关键词: 溶瘤病毒, 胰腺癌, 免疫治疗, 细胞因子, IL-21

Objective

To investigate the therapeutic potential of an interleukin-21-expressing recombinant oncolytic influenza virus (rOV-IL-21) in pancreatic cancer.

Methods

Viral replication, IL-21 expression, and cytotoxicity of rOV-IL-21 in pancreatic cancer cells were evaluated by hemagglutination assay and CCK-8 assay. A subcutaneous pancreatic cancer model was established in C57BL/6 mice, and tumor growth was monitored. Flow cytometry was used to assess intratumoral immune activation. In vivo safety was evaluated by monitoring body weight, serum ALT/AST levels, and histopathological changes in major organs. ANOVA analysis was used for comparisons among multiple groups, and Tukey multiple comparison test was used for pairwise comparisons between groups. Two-factor repeated measures analysis of variance (ANOVA) was used for comparisons involving time and treatment doses.

Results

rOV-IL-21 can stably replicate and efficiently express IL-21 in pancreatic cancer cells, exhibiting a pronounced dose-dependent cytotoxic effect. In the syngeneic subcutaneous tumor model established with KPC pancreatic cancer cells, rOV-IL-21 significantly inhibited tumor growth (P < 0.001) and prolonged the survival of mice (P = 0.001 8) . Flow cytometric analysis of tumor-infiltrating immune cells showed that rOV-IL-21 enhanced the cytotoxic function of CD8⁺ T cells, with significantly increased proportions of GzmB⁺ CD8⁺ T cells [ (19.23 ± 1.46) % vs (7.53 ± 2.28) %, P < 0.05] and reduced proportions of exhausted CD8⁺ T cells [ (3.47 ± 0.52) % vs (14.54 ± 5.91) %, P < 0.05]when compared with control groups. There were no significant differences in serum ALT or AST levels between the rOV-IL-21 and control groups (P > 0.05) .

Conclusion

rOV-IL-21 demonstrates favorable safety and potent antitumor efficacy in pancreatic cancer models, which is associated with enhanced cytotoxic function of intratumoral CD8⁺ T cells. These findings support rOV-IL-21 as a promising oncolytic virus-based immunotherapeutic strategy for pancreatic cancer.

Key words: Oncolytic virus, Pancreatic cancer, Immunotherapy, Cytokine, Interleukin-21

图1 重组溶瘤流感病毒rOV-IL-21的表征及在胰腺癌细胞中的复制及分泌IL-21情况注：a图为rOV-IL-21透射电镜扫描；b图为第4代rOV-IL-21血凝示意；c ~ d图为病毒复制折线，rOV-IL-21 （MOI = 1）感染KPC、Pan02后12、24、48、72、96 h后TCID50测定；e ~ f图为IL-21在胰腺癌细胞动态分泌折线，检测不同MOI （0，0.1，1，3，10）rOV-IL-21感染KPC、Pan02 12、24、48、72 h后上清液IL-21的浓度。^**P < 0.01

图2 rOV-IL-21体外有效抑制胰腺癌细胞活力注a ~ f图分别为CCK8法检测不同MOI （0，0.1，1，3）的rOV-IL-21感染胰腺癌细胞KPC、Panc02、PANC-1、Bxpc-3、SW1990、Aspc-1不同时间（24、48、72 h）的细胞活力，^*P < 0.05，^**P < 0.01，^***P < 0.001，^****P < 0.000 1，ns为差异无统计学意义

图3 rOV-IL-21对胰腺癌荷瘤小鼠的抗肿瘤效果及生存率影响注：a图为小鼠实验流程；b图为各组小鼠肿瘤体积生长曲线（n = 5）；c图为各组离体肿瘤组织（n = 5）；d图为各组离体肿瘤组织质量比较（n = 5）；e图为各组小鼠生存曲线（n = 5），^***P < 0.001，^****P < 0.000 1

图4 肿瘤组织流式免疫细胞分析注：末次治疗后3 d收取各组（n = 3）肿瘤组织进行流式细胞术实验，分析肿瘤组织内不同免疫细胞CD4⁺ T、Ki67⁺CD4⁺ T、IFNγ⁺ CD4⁺ T、Treg、CD8⁺ T、Ki67⁺ CD8⁺ T、IFNγ⁺ CD8⁺ T、GzmB⁺ CD8⁺ T、exhausted CD8⁺ T的比例变化。^*P < 0.05

图5 流式细胞实验检测各组肿瘤组织内exhausted CD8⁺ T细胞及GzmB⁺ CD8⁺ T细胞

图6 rOV-IL-21在小鼠胰腺癌模型的体内安全性评价注：a图为各组小鼠的体重变化折线（n = 5）；b图为各组小鼠血清ALT酶水平检测（n = 5）；c图为各组小鼠血清AST酶水平检测（n = 5）；d图为光学显微镜下观察各组小鼠心脏、肝脏、脾脏、肺脏、肾脏和脑组织的HE染色（×200），ns为差异无统计学意义

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Antitumor activity and immune activation effects of recombinant oncolytic influenza virus expressing IL-21 in pancreatic cancer

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Antitumor activity and immune activation effects of recombinant oncolytic influenza virus expressing IL-21 in pancreatic cancer