携带MART-1基因减毒产单核李斯特菌抑制小鼠恶性黑色素瘤的研究

发布时间：2018-02-21 01:25

本文关键词： 恶性黑素瘤产单核李斯特菌 MART-1　出处：《华中科技大学》2010年硕士论文　论文类型：学位论文

【摘要】：目的:探讨携带黑色素瘤分化抗原MART-1基因的减毒李斯特菌对恶性黑色素瘤的抑制作用及其机制的初步研究。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LD_(50)。C57BL/6小鼠随机分为PBS组,△inlB LM-MART-1组和△actA/△inlB LM-MART-1组。首次免疫后于第7d小鼠腹部皮下接种B16F10细胞,第14d、21d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CD~(4+)CD~(25+)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LD_(50)比LM-EGD标准株毒力分别下降2个和4个数量级。体内抑瘤试验显示△inlB LM-MART-1组和△actA/△inlB LM-MART-1组较PBS组肿瘤明显减小。与PBS组比较,△inlB LM-MART-1组抑瘤率46.95%(F=6.3,P0.05),△actA/△inlB LM-MART-1抑瘤率为83.96%( F=37.8,P0.001),差异均有统计学意义。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,各组间均有明显统计学差异(P0.05),且三组小鼠脾细胞中CD~(4+)CD~(25+)T细胞阳性率依次为2.52±0.20%,1.14±0.13%和0.44±0.15%(2.52±0.20% vs 1.14±0.13%,F=271.7,P0.001; 2.52±0.20% vs 0.44±0.15%,F=564,P0.001; 2.52±0.20% vs 0.44±0.15%,F=95.7,P0.001)。差异均有统计学意义。结论:携带MART-1基因减毒产单核李斯特菌毒性明显低于李斯特菌标准株,用重组减毒LM免疫荷瘤小鼠可以有效抑制黑色素瘤生长,并且延长小鼠生存期。
[Abstract]:Objective: to investigate the inhibitory effect of attenuated Listeria mutans carrying melanoma differentiation antigen (MART-1) gene on malignant melanoma and its mechanism. Methods: pERL3-MART-1 vector was constructed and recombinant Listeria sp. InlB LM-MART-1 and actA / inlB LM-MART-1 carrying MART-1 gene were established by electroporation. Half lethal LD_(50).C57BL/6 mice of each attenuated strain were randomly divided into PBS group by concentration gradient dilution assay. B16F10 cells were subcutaneously inoculated into the abdomen of inlB LM-MART-1 group and acta / inlB LM-MART-1 group on the 7th day after the first immunization, and repeated immunized mice on the 14th day and 21st day, respectively. The tumor size and survival status of mice were observed and recorded. The expression of MART-1 in tumor tissues was detected by real-time quantitative PCR, and the positive rate of CD~(4 T cells in spleen cells was detected by flow cytometry. Results: inlB LM-MART-1 and actA / inlB LM-MART-1.The virulence of attenuated strains inlB LM and actA / inlB LM were 2 and 4 orders of magnitude lower than those of LM-EGD standard strains, respectively. The tumor size of inlB LM-MART-1 group was significantly smaller than that of PBS group, and that of PBS group was significantly lower than that of PBS group. The tumor inhibition rate of inlB LM-MART-1 group was 46.95%, and the inhibitory rate of actA / inlB LM-MART-1 was 83.96%. The difference was statistically significant. The expression of MART-1 was increased in inlB LM-MART-1 group, inlB LM-MART-1 group and actA / inlB LM-MART-1 group. In PBS group, the positive rates of CDT cells were 1, 8.988 卤0.207 and 11.315 卤0.445, respectively. There were significant differences among the three groups (P 0.05). The positive rates of T cells in spleen cells of the three groups were 2.52 卤0.20 卤1.14 卤0.13% and 0.44 卤0.152.52 卤0.20% vs 1.14 卤0.152.52 卤0.20% vs 1.14 卤0.13F = 271.77, P 0.001respectively; 2.52 卤0.20% vs 0.44 卤0.15F564P0.001P; 2.52 卤0.20% vs 0.44 卤0.15g F95.7P 0.0011.The difference was statistically significant. Conclusion: the toxicity of Listeria monocytogenes carrying MART-1 gene is significantly lower than that of standard strains of Listeria monocytogenes. Immunizing mice with recombinant attenuated LM can effectively inhibit the growth of melanoma and prolong the survival time of mice.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.5

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