白细胞介素-24及白细胞介素-12基因治疗小鼠黑色素瘤实验研究

Experimental study of IL-24 and IL-12 gene therapy on murine melanoma in mice

[目的]研究重组质粒pEGFP-N1-白细胞介素(IL)-24基因及pGEG-IL-12基因单独应用与联合应用对小鼠黑色素瘤移植瘤的抑制作用及其机制.[方法]制备黑色素瘤B16细胞荷瘤小鼠模型,并随机分为5个组,分别向瘤体内注射给予脂质体包裹的基因重组质粒pEGFP-N1-IL-24(IL-24组),pEGFP-N1-IL-24和pGEG-IL-12(联合应用组),pGEG-IL-12(IL-12组),pEGFP-N1(空质粒组)及PBS(PBS组).每次给药前测量小鼠背部肿瘤的长径和短径,绘制肿瘤生长曲线.采用HE染色法观察各组肿瘤组织形态;RT-PCR法检测移植瘤组织中IL-24,IL-12,Bax,Bcl-2,VEGF,caspase-3及caspase-12mRNA的表达;Western Blot检测移植瘤组织中Bax,Bcl-2,VEGF,caspase-3及caspase-12蛋白的表达.[结果]肿瘤生长曲线观察结果表明,IL-24组、IL-12组及联合应用组肿瘤体积均明显小于空质粒组和PBS组(P<0.05),联合应用组肿瘤体积最小(P<0.05).HE染色结果显示,空质粒组多数细胞生长差,形态不规则,细胞皱缩,结构稀疏,细胞核溶解、变形,细胞成片坏死.RT-PCR检测结果表明,IL-24组和IL-12组分别有IL-24和IL-12基因mRNA表达,联合应用组同时有IL-24和IL-12基因mRNA表达,而空质粒组和PBS组均未见表达;IL-24组、IL-12组及联合应用组Bax基因mRNA表达水平均明显高于空质粒组和PBS组(P<0.05),联合应用组Bax基因mRNA的表达水平最高,而Bcl-2和VEGF基因mRNA的表达水平则显著降低(P<0.05).Western Blot检测结果表明,IL-24组、IL-12组及联合应用组Bax,caspase-3,caspase-12蛋白的表达水平均明显高于空质粒组和PBS组(P<0.05),联合应用组中Bax,caspase-3,caspase-12蛋白的表达水平最高,而Bcl-2和VEGF蛋白的表达水平则显著降低(P<0.05).[结论]IL-24和IL-12对荷瘤小鼠有治疗作用,二者联合应用治疗效果更强.

OBJECTIVE To study the inhibitory activity and mechanism of recombinant plasmid pEGFP-N1-IL-24 gene and pGEG-IL-12 gene single application and combined application to melanoma B16 cells in mice xenografts.METHODS The tumor-bearing mice models with melanoma B16 cells were established and randomly divided into 5groups,and it was respectively injected into the tumors that the recombinant plasmid liposome of pEGFP-N1-IL-24（IL-24 group）,pEGFP-N1-IL-24 and pGEG-IL-12（combined application group）,pGEG-IL-12（IL-12group）,pEGFP-N1（empty plasmid group）and PBS buffer solution（PBS group）.The long and short diameter of transplanted tumor was measured before injection to draw the growth curve of tumor.The histomorphology of transplanted tumors were observed by HE staining.Expressions of mRNA of IL-24,IL-12,Bax,Bcl-2 and VEGF in transplanted tumor were detected by RT-PCR,and protein expressions of Bax,Bcl-2,VEGF,caspase-3and caspase-12 in transplanted tumors were detected by Western blot.RESULTSThe gross tumor volume was significantly smaller in the IL-24 and IL-12 groups than in the empty plasmid and PBS groups（P〈0.05）,and was smallest in the combined application group（P〈0.05）.The most cells in the recombinant plasmid group were poor growth,irregular shape,cell shrinkage,sparse,karyolysis,deformation,patches of necrosis.The expressions of mRNA of IL-24 and IL-12 were in the IL-24 and IL-12 groups,and in the combined application group,respectively,and were not in the empty plasmid and PBS groups,and the expression of mRNA of Bax was significantly higher in the IL-24,IL-12 and combined application groups than in the empty plasmid and PBS groups（P〈0.05）,and was the highest in the combined application group,but the expressions of mRNA of Bcl-2and VEGF were on the contrary（P〈0.05）.The protein expressions of Bax,caspase-3and caspase-12 were significantly higher in the combined application group than in the empty plasmid and PBS groups（P〈0.05）,and the highest in the combined application group,but the protein expressions of Bcl-2and VEGF were on the contrary（P〈0.05）.CONCLUSIONIL-24 and IL-12 have inhibitory effect to the mice B16 melanoma growth,and the treatment effect of combined application with IL-24 and IL-12 is stronger.