靶向VEGF基因的siRNA联合树突状细胞介导的CTL对MCF-7细胞作用的研究

Effects of siRNA directed against VEGF gene and CTL mediated by DCs loaded tumor antigen on MCF-7 Breast Cancer cells

目的探讨靶向血管内皮细胞生长因子(VEGF)基因的小干扰RNA(siRNA)联合负载肿瘤抗原的树突状细胞(DC)致敏的细胞毒性T淋巴细胞(CTL)的联合抗瘤作用。方法体外应用靶向VEGF基因最佳转染浓度(100nmol/L,增加浓度不再提高沉默作用)的siRNA联合负载人乳腺癌细胞(MCF-7细胞)抗原的DC介导的CTL(siRNAVEGF+CTL组)共同作用于MCF-7细胞,同时设立空白对照组和空白+CTL组(只加无血清无抗生素培养基);脂质体组和脂质体+CTL组(只加LipofectamineTM2000);siRNAVEGF-组(100nmol/LsiRNA);siRNASCR组和siR-NASCR+CTL组(100nmol/LsiRNASCR),每组做6个平行孔,四甲基偶氮唑蓝(MTT)法检测siRNAVEGF+CTL组和各对照组肿瘤杀伤活性(n=6),Hoechst33258核染色观察细胞的凋亡(n=3)。结果siRNAVEGF+CTL组、siR-NAVEGF-组siRNASCRCTL组肿瘤杀伤活性分别为99.37%,51.17%和43.94%,siRNAVEGF+CTL组与对照组比较可明显杀伤肿瘤细胞,瘤细胞几乎完全溶解,Hoechst33258显示细胞核呈明显的细胞凋亡改变。结论体外实验显示靶向VEGF的siRNA联合负载肿瘤抗原DC致敏的CTL能有效抑制乳腺癌MCF-7细胞的生长,二者的联合应用抗瘤效果显著。

Objective To investigate the effects of siRNA directed against VEGF gene and cytotoxic T lymphocyte（CTL）mediated by DCs loaded tumor-specific antigen on MCF-7 Breast Cancer cells.Medhods In vitro small interfering RNAs（siRNAs）directed against VEGF gene were designed and transfected into MCF-7 breast cancer cells at the optimal concentration（100 nmol/L）using cationic liposome Lipofectamine^TM 2000,then combinted CTL mediated by DCs（siRNAVEGF＋CTL group）were used to against MCF-7 breast cancer cells.At the same time,the following groups were designed：blank control group and blank＋CTL group（only with serum-free medium without antibiotics）;liposome group and liposome＋CTL group（added Lipofectamine^TM2000 only）;siRNAVEGF-group and siRNAVEGF＋CTL group（100 nmol/L siRNA）;siRNASCR and siRNASCR＋CTL group（100 nmol/L siRNASCR）.Tumor killing effects in both siRNAVEGF＋CTL group and control groups were measured by MTT（n=6）assay and Hoechst33258（n=3）.Results The tumor killing effects in siRNAVEGF＋CTL,siRNAVEGF and siRNASCR CTL groups were 99.37%,51.17% and 43.94%,respectively,obviously increased in siRNAVEGF＋CTL group compared with the control groups（P〈0.01）.The tumor cells were almost completely lysis and Hoechst33258 showed obviously apoptosis in nucleis in siRNAVEGF＋CTL group.Conclusion The experiment demonstrated that siRNA directed against VEGF gene combined CTL mediated by DCs could inhibit the growth of MCF-7 cells,and with a more significant antitumor effect by the combined application.