通讯作者: 张林, stepinghuns2@163.com

DOI：10.12201/bmr.202607.00010

Preparation of a novel recombinant vaccinia virus carrying CXCL9 and IL-7 genes and its anti-tumor effect in vitro

• 摘要：目的：通过分子生物学方法制备携带CXCL9和IL-7双基因的新型重组溶瘤痘苗病毒并通过体外细胞学实验初验证该重组溶瘤病毒感染、杀伤胃癌细胞的作用。方法：首先分步构建携带CXCL9和IL-7双基因的重组痘苗病毒真核表达质粒pSC65-CXCL9-IL7，继而以该载体真核细胞内重组痘苗病毒株，经过有限稀释法反复筛选纯化后,获得纯化的重组溶瘤痘苗病毒VV- CXCL9-IL7，而后经感染HeLa细胞扩大培养并采用氯化铯梯度离心法浓缩纯化后获得高滴度的重组溶瘤痘病毒。以此重组溶瘤痘病毒体外感染人胃癌细胞系SGC7901细胞，通过半数细胞培养物感染量TCID50法检测重组病毒感染能力，用ELISA 法检测细胞培养上清液中CXCL9和IL-7的浓度。采用MTT法检测VV-CXCL9-IL7对SGC7901细胞的杀伤力。采用细胞生长曲线法检测重组溶瘤痘病毒感染对细胞生长的影响，采用流式细胞仪法检测被感染SGC7901细胞的细胞周期及凋亡情况变化。结果：成功构建了重组溶瘤痘病毒VV-CXCL9-IL7，通过上清液浓度检测初步验证了IL-7和CXCL9趋化因子产生及释放作用。细胞感染干预结果显示VV-CXCL9-IL7重组溶瘤痘病毒干预组肿瘤细胞杀伤作用明显强于各对照组且肿瘤生长明显减缓，显著慢于其它各对照组，溶瘤痘病毒干预组细胞G0/G1期比率显著高于各对照组其凋亡率明显增加。结论：在体外细胞学干预实验中采用VV-CXCL9-IL7重组溶瘤病毒感染干预胃癌细胞具有较强的抑瘤作用，对于胃癌这种可局部内镜下直接局部给药形成感染的肿瘤具有潜在的临床应用价值。

  关键词： 胃癌; 溶瘤痘苗病毒; CXCL9; IL-7; 生物免疫治疗

   

  Abstract: Objective: To prepare a novel recombinant vaccinia virus carrying CXCL9 and IL-7 double genes by molecular biological method, and to preliminarily verify the effect of the recombinant vaccinia virus in infecting and killing gastric cancer cells by in vitro cytological experiments. Methods: The recombinant vaccinia virus eukaryotic expression plasmid pSC65-CXCL9-IL7 carrying CXCL9 and IL-7 double genes was constructed step by step, and then the vaccinia virus strain was recombined in eukaryotic cells with this vector. After repeated screening and purification with limited dilution method, purified recombinant oncolytic poxvirus VV- CXCL9-IL7 was obtained, Then, the recombinant oncolytic poxvirus with high titer was obtained by infecting HeLa cells to expand the culture and using cesium chloride gradient centrifugation to concentrate and purify it. The recombinant oncolytic poxvirus was used to infect human gastric cancer cell line SGC7901 in vitro. The infectivity of recombinant virus was detected by TCID50 method, and the concentration of CXCL9 and IL-7 in cell culture supernatant was detected by ELISA. The cytotoxicity of VV-CXCL9-IL7 to SGC7901 cells was detected by MTT method. The effect of recombinant oncolytic poxvirus infection on cell growth was detected by cell growth curve method, and the changes of cell cycle and apoptosis of infected SGC7901 cells were detected by flow cytometry. Results: The recombinant oncolytic poxvirus VV-CXCL9-IL7 was successfully constructed, and the production and release of chemokines IL-7 and CXCL9 were preliminarily verified by the detection of the concentration of supernatant. The results of cell infection intervention showed that the killing effect of tumor cells in the VV-CXCL9-IL7 recombinant oncolytic poxvirus intervention group was significantly stronger than that in each control group, and the tumor growth was significantly slower than that in other control groups. The G0/G1 phase ratio of cells in the oncolytic poxvirus intervention group was significantly higher than that in each control group, and its apoptosis rate was significantly increased. Conclusion: In vitro cytological intervention experiment, VV-CXCL9-IL7 recombinant infection has a strong anti-tumor effect on gastric cancer cells, and has potential clinical application value for gastric cancer, which can be infected by local direct drug administration.

  Key words: gastric cancer; Oncolytic poxvirus; CXCL9; IL-7; Bioimmunotherapy

  提交时间：2026-08-26

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• 序号	提交日期	编号	操作
  1	2026-07-03	10.12201/bmr.202607.00010V1	下载

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