仙台病毒囊膜抗小鼠黑色素瘤的研究

发布时间：2018-01-28 19:30

本文关键词： 仙台病毒囊膜小鼠恶性黑色素瘤黑色素瘤细胞树突状细胞凋亡　出处：《扬州大学》2011年硕士论文　论文类型：学位论文

【摘要】：小鼠恶性黑色素瘤(Malignant melanoma, MM)具有恶性程度高、易转移和易复发等特点,目前常规疗法对其治疗效果不是很好。研究表明,仙台病毒囊膜(Hemagglutinating virus of Japan Envelope, HVJ-E)具备强大的抑制或消除肿瘤的功能,且在动物肿瘤模型上得到了验证,但目前尚未有直接用HVJ-E治疗小鼠黑色素瘤的研究报道。因此,我们开展用HVJ-E治疗小鼠黑色素瘤的研究,旨在初步探讨其抗瘤机制。首先,通过体外实验观察小鼠黑色素瘤细胞(B16F10)经HVJ-E作用后的形态学变化,MTT、流式细胞检测、免疫组化、免疫印迹法检测经HVJ-E作用后的B16F10细胞的增殖活性、凋亡水平及IGF1R α、β-catenin表达情况。倒置显微镜观察细胞形态结果显示,PBS阴性对照组细胞贴壁良好,呈梭形,生长活跃,折光性好；而HVJ-E处理组细胞皱缩,破裂,呈不规则形态,有的细胞两端出现细长的伪足,脱落细胞增多。MTT法检测结果显示,不同浓度的HVJ-E对细胞都有明显抑制作用并呈剂量-效应关系,500MOI HVJ-E组在处理72h后与同时段对照组相比有显著差异(p0.05)；1000MOI HVJ-E组在处理72h后与同时段对照组相比差异极显著(p0.01)；随着HVJ-E处理浓度的增大,B16F10细胞的凋亡率升高；HVJ-E组的IGF1Rα、β-catenin的表达明显低于PBS对照组,且随着HVJ-E处理浓度的增大,IGFlRα、β-catenin蛋白表达量下降。该实验说明HVJ-E具有直接促使小鼠黑色素瘤细胞凋亡的作用。其次,通过体外分离培养小鼠骨髓树突状细胞(dendritic cell, DC),以500MOIHVJ-E病毒样粒子刺激小鼠骨髓DC,同时设PBS阴性对照与LPS阳性对照,培养48h后,检测各组DC细胞上清中细胞因子的分泌水平和DC细胞表面分子的表达率。结果显示,HVJ-E处理组DC细胞分泌的IFN-β、IFN-γ水平显著高于PBS、LPS对照组(p0.05),IL-6、TNF-α水平显著高于PBS对照组(p0.05),但略低于LPS对照组；HVJ-E处理组的DC细胞表面分子CD80、CD40、CDllc和MHCⅡ均呈高表达。该实验说明HVJ-E能够刺激未成熟DC成熟并且分泌细胞因子从而发挥抗瘤作用。最后,通过C57BL/6J小鼠皮内接种B16F10细胞制作小鼠黑色素瘤模型,待瘤体直径达0.5cm时,将荷瘤小鼠随机分组,每组20只,实验组小鼠瘤内注射HVJ-E病毒样粒子,同时设PBS阴性对照组,4天之后重复治疗,连续治疗3次,每次治疗后测量肿瘤体积,观察并分析荷瘤鼠的存活率。另取两组荷瘤小鼠,每组6只,瘤内注射HVJ-E病毒样粒子,同时设PBS阴性对照,连续注射3天,最后1次注射后24,48,120h时,每组各处死2只小鼠,分别取瘤组织进行荧光定量PCR检测CD4, CD8, CXCL10, CDllc, IL-2, MITF, IGFIRα, β-catenin mRNA的转录水平。治疗结果表明,HVJ-E治疗组小鼠的肿瘤体积显著小于PBS对照组,且在成瘤后40天内,HVJ-E治疗组小鼠的存活率为80%,而对照组为0%。荧光定量PCR结果显示HVJ-E治疗组CD4, CD8, CXCL10, CDllc, IL-2mRNA转录水平在24,48,120h时均高于PBS对照组,MITF, IGFIRα,β-catenin mRNA转录水平在24,48h时均低于PBS对照组。该实验说明HVJ-E能激发机体的特异性与非特异性细胞免疫,从而延长黑色素瘤小鼠的生存期。体内外实验证明仙台病毒囊膜(HVJ-E)能刺激DC成熟,释放细胞因子,并具备潜在的免疫增强效应；HVJ-E能直接诱导小鼠黑色素瘤细胞凋亡,其作用机制可能与MITF, IGF1R α和β-catenin的表达下降有关；HVJ-E直接进行瘤内注射后能显著抑制小鼠恶性黑色素瘤的生长,延长荷瘤小鼠的生存期,其作用机制不仅仅体现在HVJ-E可诱导特异性细胞免疫的产生,还可能是HVJ-E的致肿瘤细胞凋亡效应,但具体的作用机制仍需进一步的研究。可以肯定的是,HVJ-E可有效减缓肿瘤的生长和延长荷瘤鼠生存期,并有望为黑色素瘤治疗提供新的理论依据和方法。
[Abstract]:The malignant melanoma ( MM ) of mice has the characteristics of high malignancy degree , easy metastasis and easy recurrence . The study shows that Hemaglobating virus of Japan Envelope ( HVJ - E ) has the function of inhibiting or eliminating the tumor and has been verified on animal tumor model . However , it has not been reported directly by HVJ - E in the treatment of mouse melanoma . Therefore , we have carried out the study of treating mouse melanoma with HVJ - E , aiming at initially exploring its anti - tumor mechanism . The expression of IGF1R 伪 , 尾 - catenin in HVJ - E group was significantly lower than that in control group ( P < 0.01 ) . The results showed that the levels of IFN - 尾 and IFN - 纬 were significantly higher in HVJ - E treated group than in PBS , LPS control group ( P < 0.05 ) , IL - 6 and TNF - 伪 were significantly higher than that in PBS control group ( P < 0.05 ) . The results showed that HVJ - E could stimulate immature DC maturation and secrete cytokines to exert anti - tumor effect . The results showed that HVJ - E could stimulate the specific non - specific cell immunity of mice and prolong the survival time of melanoma mice . HVJ - E can significantly inhibit the growth of mouse melanoma cells and prolong the survival of tumor - bearing mice . The mechanism of HVJ - E can significantly inhibit the growth of mouse malignant melanoma and prolong the survival time of tumor - bearing mice .

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.5

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