本文關(guān)鍵詞： 惡性黑素瘤 產(chǎn)單核李斯特菌 MART-1　出處：《華中科技大學(xué)》2010年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:探討攜帶黑色素瘤分化抗原MART-1基因的減毒李斯特菌對(duì)惡性黑色素瘤的抑制作用及其機(jī)制的初步研究。 方法:構(gòu)建pERL3-MART-1載體,通過(guò)電轉(zhuǎn)化建立攜帶MART-1基因的△inlB LM-MART-1和△actA/△inlB LM-MART-1重組李斯特菌。濃度梯度稀釋法測(cè)定各減毒菌株半數(shù)致死量LD_(50)。C57BL/6小鼠隨機(jī)分為PBS組,△inlB LM-MART-1組和△actA/△inlB LM-MART-1組。首次免疫后于第7d小鼠腹部皮下接種B16F10細(xì)胞,第14d、21d重復(fù)免疫小鼠,觀(guān)察并記錄腫瘤大小及小鼠生存狀態(tài)。應(yīng)用實(shí)時(shí)定量PCR檢測(cè)腫瘤組織中MART-1表達(dá)量。流式細(xì)胞術(shù)檢測(cè)小鼠脾細(xì)胞中CD~(4+)CD~(25+)T細(xì)胞陽(yáng)性率。 結(jié)果:經(jīng)鑒定成功構(gòu)建△inlB LM-MART-1和△actA/△inlB LM-MART-1。減毒株△inlB LM和△actA/△inlB LM的LD_(50)比LM-EGD標(biāo)準(zhǔn)株毒力分別下降2個(gè)和4個(gè)數(shù)量級(jí)。體內(nèi)抑瘤試驗(yàn)顯示△inlB LM-MART-1組和△actA/△inlB LM-MART-1組較PBS組腫瘤明顯減小。與PBS組比較,△inlB LM-MART-1組抑瘤率46.95%(F=6.3,P0.05),△actA/△inlB LM-MART-1抑瘤率為83.96%( F=37.8,P0.001),差異均有統(tǒng)計(jì)學(xué)意義。PBS組、△inlB LM-MART-1組和△actA/△inlB LM-MART-1組腫瘤組織中MART-1表達(dá)量遞增,以PBS組表達(dá)量為1,后兩組為8.988±0.207和11.315±0.445,各組間均有明顯統(tǒng)計(jì)學(xué)差異(P0.05),且三組小鼠脾細(xì)胞中CD~(4+)CD~(25+)T細(xì)胞陽(yáng)性率依次為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)。差異均有統(tǒng)計(jì)學(xué)意義。 結(jié)論:攜帶MART-1基因減毒產(chǎn)單核李斯特菌毒性明顯低于李斯特菌標(biāo)準(zhǔn)株,用重組減毒LM免疫荷瘤小鼠可以有效抑制黑色素瘤生長(zhǎng),并且延長(zhǎng)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 鹵0.207 and 11.315 鹵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 鹵0.20 鹵1.14 鹵0.13% and 0.44 鹵0.152.52 鹵0.20% vs 1.14 鹵0.152.52 鹵0.20% vs 1.14 鹵0.13F = 271.77, P 0.001respectively; 2.52 鹵0.20% vs 0.44 鹵0.15F564P0.001P; 2.52 鹵0.20% vs 0.44 鹵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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R739.5

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