慢病毒介导人白细胞介素12转染鼠骨髓间充质干细胞可抑制CT26瘤体的生长

Lentivirus-mediated human interleukin-12 fusion gene-transfected rat bone marrow mesenchymal stem cells inhibit CT26 tumor growth

背景:白细胞介素12作为最具潜力的抑瘤因子一直是研究的热点,但其在结直肠癌方面的研究相对较少。目的:构建稳定表达人白细胞介素12的大鼠骨髓间充质干细胞株(h IL-12-BMSCs),观察其对大鼠结肠癌模型的影响。方法:设计并合成引物,PCR扩增并回收纯化后的p40和p35基因片段,行Overlap-ping PCR连接获得h IL-12的单链双亚基表达融合基因,与慢病毒包装系统共转染293T细胞,构建慢病毒过表达重组体,再转染大鼠骨髓间充质干细胞,药物筛选培育出稳转株。32只裸鼠皮下注射CT26细胞悬液造模成功后,随机分为4组:瘤周分别注射0.2 m L h IL-12-BMSCs(h IL-12-BMSCs组)、0.2 m L PBS溶液(PBS组)、0.2 m L骨髓间充质干细胞(骨髓间充质干细胞组)、0.2 m L表达IL-12的慢病毒液(LV-IL-12组),统计并分析各组瘤体生长情况。结果与结论:(1)观察期内PBS组与骨髓间充质干细胞组肿瘤体积差异无显著性意义(P>0.05),表明骨髓间充质干细胞本身对CT26瘤体生长无明显促进或抑制作用;(2)注射7 d后h IL-12-BMSCs组与PBS组、PBS组与LV-IL-12组间肿瘤体积差异均有显著性意义(P<0.01),表明白细胞介素12对CT26瘤体生长有明显抑制作用;(3)注射10 d后h IL-12-BMSCs组与LV-IL-12组间肿瘤体积差异有显著性意义(P<0.05),表明h IL-12-BMSCs抑制CT26生长优于LV-IL-12;(4)结果表明,h IL-12-BMSCs对裸鼠CT26移植瘤体生长有明显的抑制作用。

BACKGROUND:Interleukin-12 as the most potent anti-tumor factor has been a hot topic in research,but little is reported on its use in the treatment of colorectal cancer.OBJECTIVE:To construct rat bone marrow mesenchymal stem cell lines stably expressing human interleukin-12(hIL-12-BMSCs),and to observe its effect on colorectal cancer(CT26).METHODS:Primers were designed and synthesized,and purified p40 and p35 gene fragments were amplified by PCR.The single-chain double-subunit fusion gene,hIL-12,was obtained by Overlap-ping PCR ligation,and 293T cells were co-transfected with lentiviral packaging system.Recombinant lenovirus overexpressing hIL-12(LV-IL-12)was constructed to transfect rat BMSCs.The stable strain was then cultured via drug screening.Thirty-two nude mice were subcutaneously injected with CT26 cells to make animal models,and then randomly divided into four groups to receive peritumoral injection of hIL-12-BMSCs(0.2 mL;hIL-12-BMSCs group),0.2 mL of PBS(PBS group),0.2 mL of BMSCs(BMSCs group),or 0.2 mL of LV-IL-12(LV-IL-12 group).Tumor growth was then statistically analyzed in each group.RESULTS AND CONCLUSION:There was no significant difference in tumor volume between the PBS and BMSCs groups(P>0.05),indicating that BMSCs cannot promote or inhibit the growth of CT26 tumors.However,from the 7th day after injection,the tumor volume showed a significant difference between hIL-12-BMSCs and PBS groups as well as between PBS and LV-IL-12 groups(P<0.01),indicating that Hil-12 has a significant inhibitory effect on CT26 tumor growth.From the 10th day after injection,a significant difference in the tumor volume was found between the hIL-12-BMSCs and LV-IL-12 groups(P<0.05),indicating that hIL-12-BMSCs are more effective than LV-IL-12 in inhibiting the growth of CT26.To conclude,hIL-12-BMSCs considerably inhibit the growth of CT26 tumor.