携带MART-1基因的减毒单核细胞增多性李斯特菌抑制小鼠恶性黑素瘤的研究

目的 探讨携带黑素瘤分化抗原MART-1基因的减毒单核细胞增多性李斯特菌（LM）对恶性黑素瘤的抑制作用及其机制。方法 构建pERL3-MART-1载体，通过电转化建立携带MART-1基因的△inlB LM-MART-1和△actA/△inlB LM-MART-1重组李斯特菌。浓度梯度稀释法测定各减毒菌株半数致死量（LD50）。C57BL/6小鼠随机分为磷酸盐缓冲液（PBS）组，△inlB LM- MART-1组和△actA/△inlB LM- MART-1组。首次免疫后第7天于小鼠腹部皮下接种B16F10细胞，第14天、21天重复免疫小鼠，观察并记录肿瘤大小及小鼠生存状态。应用实时定量PCR检测肿瘤组织中MART-1表达量。流式细胞术检测小鼠脾细胞中CD4+CD25+ T细胞阳性率。结果 经鉴定成功构建△inlB LM-MART-1和△actA/△inlB LM-MART-1。减毒株△inlB LM 和△actA/△inlB LM的LD50比LM-EGDe标准株毒力分别下降100倍和10 000倍。体内抑瘤试验显示，与PBS组比较，△inlB LM- MART-1组抑瘤率为46.95%，△actA/△inlB LM-MART-1抑瘤率为83.96%，差异均有统计学意义（P < 0.05和0.01）。PBS组、△inlB LM-MART-1组和△actA/△inlB LM-MART-1组肿瘤组织中MART-1表达量递增，以ＰＢＳ组表达量为１，后两组为8.988 ± 0.207和11.315 ± 0.445，各组间差异均有统计学意义（P值均 < 0.05），且三组小鼠脾细胞中CD4+CD25+ T细胞阳性率依次为（2.52 ± 0.20）%、（1.14 ± 0.13）%和（0.44 ± 0.15）%，组间差异均有统计学意义（P值 < 0.01）。结论 携带MART-1基因的减毒LM毒性明显低于LM标准株，而且可以有效抑制黑素瘤生长，延长小鼠生存期。

Inhibitory effect of live-attenuated Listeria monocytogenes-based vaccines carrying MART-1 gene on mouse malignant melanoma

Objective To investigate the inhibitory effect of live-attenuated Listeria monocytogenes (LM)-based vaccines expressing the gene encoding a melanoma differentiation antigen,MART-1,on malignant melanoma and their mechanism.Methods The constructed plasmid pERL3-MART-1 was used to transform live-attenuated LM by electroporation to construct recombinant LM.i.e.△inlB LM-MART-1 and △actA/△inlB LM-MART-1.The half lethal dose (LD50) of attenuated listeria strains was determined by concentration gradient dilution method.C57BL/6 mice were randomly divided into three groups,namely PBS group,△inlB LM-MART-1 group and △actA/△inlB LM-MART-1 group.Mice were inoculated by intraperitoneal injection of O.1 LD50 of each rLM strain or PBS only.One week later,the mice were injected subcutaneously with 1×105 B16F10 cells(a mouse melanoma cell strain)in 200μl of PBS.Reimmunization was performed on day 14 and 21.Subsequently,the growth of tumor and survival of tumor bearing mice were observed.All mice were killed on day 28,and tumor tissue as well as splenocytes were obtained from these mice for the detection of MART-1 gene expression by real-time quantitative PCR and the percentage of CD4+CD25+T cell by flow cytometry.Results The recombinant △inlB LM-MART-1 and △actA/△inlB LM-MART-1 were constructed successfully.The LD50 of △inlB LM and △actA/△inlB LM was lower than LM-EGDe by 100 and 10 000 times respectively.Compared with PBS,the tumor growth was inhibited with △inlB LM-MART-1 by 46.95%(F=6.3,P<0.05),and by 83.96% with △actA/△inlB LM-MART-1(F=37.8,P<0.01).The relative expression level of MART-1 in △inlB LM-MART-1 group and △actA/△inlB LM-MART-1 group was 8.988±0.207 and 11.315±0.445 times that in PBS group (both P<0.05).The percentage of CD4+CD25+T cells in splenocytes was (2.52±0.20)%,(1.14±0.13)% and (0.44±0.15)% in PBS group,△ialB LM-MART-1 group and △actA/△inlB LM-MART-1 group,respectively;the differences were statistically significant between the three groups (all P