仙台病毒Tianjin株缺损干扰颗粒对荷瘤小鼠的抑瘤作用

Anti-tumor effect of Sendai virus Tianjin strain defective interfering particles on tumor-bearing mice

目的通过小鼠结肠癌动物模型探讨仙台病毒Tianjin株缺损干扰颗粒（DIP）的抗肿瘤作用及其机制。方法将CT26细胞悬液0．1ml皮下注射BalB／c小鼠背部，建立小鼠结肠癌模型，将小鼠随机分为Tianjin株DIP组（注射CT26后第4、7、10和13天，每天瘤内注射1次Tianjin株DIP0．1m1）和生理盐水对照组（瘤内注射生理盐水0．1ml），每组10只。通过测量肿瘤大小和观察小鼠存活率确定DIP的抑瘤作用。采用流式细胞仪和酶联免疫吸附（ELISA）法检测DIP在体外对小鼠树突状细胞（DCs）成熟及分泌白细胞介素6（IL-6）、干扰素Q（IFN-α）和肿瘤坏死因子α（TNF-α）的促进作用。采用realtimeRT—PCR和免疫组化法检测DIP对小鼠肿瘤组织中CD4＋、CD8＋T细胞及CD11c＋DCs表达的影响。结果至注射CT26后第22天，Tianjin株DIP组小鼠的肿瘤体积为（33．2±2．0）mm3，与生理盐水对照组[（2376．0±130．8）mm3]比较，差异有统计学意义（P〈0．01）。至注射CT26后第50天，Tianjin株DIP组小鼠的存活率为90．0％，明显高于生理盐水对照组（30．0％，P〈0．01）。流式细胞仪检测显示，小鼠DCs表面CIM0、CD80和CD86标志分子的表达率随病毒剂量的升高而升高，且Tianjin株DIP组和完整病毒组之间的差异无统计学意义（均P〉0．05）。ELISA法检测显示，DIP能够促进小鼠DCs分泌IL-6、IFN-α和TNF-α，且呈剂量依赖性。realtimeRT-PCR检测结果显示，停止给药后24、48和120h取得的小鼠肿瘤组织中CD4、CD8和CDllcmRNA的表达水平均有所升高，且在120h表达达高峰。免疫组化检测结果显示，Tianjin株DIP组肿瘤组织中CD4＋T细胞、CD8＋T细胞和CD11c＋DCs占总细胞的百分比分别为（21．60±1．49）％、（22．12±2．84）％和（23．05±2．91）％，而生理盐水对照组分别为（2．62±0．60）％、（4．05±0．12）％和（3．10±0．09）％，差异均有统计学意义（均P〈0．05）。结论Tianjin株DIP可以在小鼠体内发挥抗肿瘤作用，其作用机制与激活DCs和T细胞诱导的抗肿瘤免疫有关。

Objective To explore the anti-tumor effect and its mechanism of Sendai virus Tianjin strain defective interfering particles （DIP） on mouse models of colon carcinoma. Methods CT26 cells （5 × 10^6/0. 1 ml） were subcutaneously injected into the back of Bal B/c mice to establish routine colon carcinoma model. After the tumors reached 5 mm in diameter, the mice were randomly divided into Tianjin strain DIP group and saline control group. The former was intratumorally injected with Tianjin strain DIP （0. 1 ml） once a day on day 4, 7, 10 and 13 after CT26 cell inoculation. The latter was intratumorally injected with the same volume of saline. Tumor volume and survival rate of the mice were calculated to confirm the anti-tumor effect of DIP. Flow cytometry and ELISA were used to examine the maturation and release of cytokines IL-6, IFN-α and TNF-α from murine myeloid dendritic cells （DCs） induced by Tianjin strain DIP. Moreover, real-time RT-PCR and immunohistochemistry were performed to identify whether the Tianjin strain DIP could induce infiltration of CD11 c ＋ DCs, CD4 ＋ and CD8 ＋ T ceils in the tumors. Results On day 22 after CT26 cell inoculation, the average tumor volume of the Tianjin strain DIP group was （33.2 ＋ 2. O） mm3 , significantly smaller than that of the control group [ （2 376.0 ± 130.8） mm3 , P 〈 0.01 ]. On day 50 after CT26 cell inoculation, the survival rate of mice was 90.0% in the Tianjin strain DIP group, much higher than that of the control group （30.0% , P 〈0.01 ）. Flow cytometry analysis showed that the expression of markers of DCs maturation, including CD40, CD80 and CD86, was dose-dependently increased by DIP or intact virus. No statistically significant difference was found betweent the DIP and intact virus groups. ELISA results showed that DIP could stimulate the secretion of IL-6, IFN-α and TNF-α from mouse DCs. The secretion of all of the cytokines was dose-dependently increased by DIP or intact virus. Real-time RT-PCR revealed that the expression of CD4, CD8 and CD11c mRNAs was increased in tumors treated with DIP compared with that of the saline group at all time points. Moreover, the expression level of all of them remained maximal at 120 h after the last treatment. Immunohistochemical staining revealed that the ratios of CD4＋, CD8＋ T cells or CDllc＋ DCs to total cells were （21.60±1.49）%, （22.12 ± 2.84） % and （23.05 ± 2.91 ） %, respectively, in the DIP-treated tumors. In the tumors treated by saline, the ratios were （2.62 ± 0.60） %, （4.05 ± 0. 12 ） % and （ 3.10 ± 0.09） %, respectively. The difference between experimental group and control group had statistical significance. Conclusions Tianjin strain DIP may exert anti-tumor effect on tumor-bearing mice. The mechanism is related with the antitumor immunity induced by DCs and T cells.