恩度联合mut1抗原特异DC-T细胞对Lewis肺癌小鼠的抗肿瘤效应研究

发布时间：2018-05-20 00:16

本文选题：肺癌 + 恩度　；参考：《山东大学》2015年硕士论文

【摘要】：目的肿瘤的发生、侵袭、转移都与肿瘤的的血管新生密不可分,因此抑制肿瘤的血管新生是控制肿瘤生长的新热点。重组人血管内皮抑素(恩度,Endostar)是通过抑制血管生长起抗肿瘤作用的一种药物。研究证实单独应用抗血管新生药物不能带来长期的生存获益,而联合细胞免疫治疗可能获得持续性的肿瘤消退,这提示抗血管新生药物与细胞免疫治疗有协同效应。树突状细胞(dendritic cells,DCs)是功能最强的专职抗原递呈细胞(antigen-presenting cells, APCs),在调控机体固有和适应性免疫应答中起重要的作用,负载肿瘤抗原的树突状细胞是近年来被广泛研究生物治疗之一。本实验通过C57BL/6小鼠Lewis肺癌荷瘤模型,研究恩度联合负载mut1抗原特异的DC-T细胞的抗肿瘤效应及对肿瘤微环境内免疫网络的影响,进一步明确恩度联合细胞免疫治疗的抗肿瘤效应,从而为开展抗血管新生的分子靶向治疗与细胞免疫治疗的联合提供实验基础和理论依据。方法1.培养Lewis肺癌细胞：显微镜下观察细胞状态和数量,及时换液和传代,待细胞生长进入对数期后,用于实验。2.制备负载mut1抗原多肽的DC-T细胞：分离及培养小鼠脾T淋巴细胞,分离及培养小鼠来源的树突状细胞,负载mut1抗原多肽的树突状细胞与T淋巴细胞共培养制备mut1抗原特异性的DC-T细胞。3.构建C57BL/6小鼠Lewis肺癌皮下种植瘤模型并分组：将C57BL/6小鼠荷瘤后随机分为A组(PBS对照组)、B组(DC-T组)及C组(DC-T+恩度组)。荷瘤后第7天开始给予治疗干预：A组给予尾静脉注射PBS溶液,B组及C组给予尾静脉输注负载mutl抗原肽DC-T细胞,C组给予尾静脉注射恩度(重组人血管内皮抑素)。4.肿瘤生长情况及抑瘤率的测定：荷瘤后隔日测量各组小鼠体质量及移植瘤的最大长径和短径,计算肿瘤体积并绘制生长曲线；末次干预24小时后处死小鼠,解剖出肿瘤组织称其体质量并计算抑瘤率。5.免疫组化SP法检测各组肿瘤组织微血管密度(MVD)。6.Western blotting检测各组肿瘤组织中VEGF-A 和 HIF-1α的表达。7.流式细胞术检测肿瘤组织中MDSC、TAM (M1/M2)、mDC和CD8+T细胞的比例。结果1.与A组相比,B组及C组小鼠体质量及肿瘤体积增长缓慢,B组肿瘤质量小于A组,差异具有统计学意义(P0.05),抑瘤率为25%；C组肿瘤质量明显小于A组,差异具有统计学意义(P0.01),抑瘤率为52%。2.MVD计数统计结果示B组与A组相比MVD计数减少,差异具有统计学意义(P0.05)；C组与A组相比MVD计数明显减少,差异具有统计学意义(P0.01)。3.Western blotting结果显示,与A组相比,B组(P0.05)及C组(P0.01)肿瘤组织内的VEGF-A蛋白表达水平下降,HIF-1 α蛋白表达水平增加4.流式细胞术分析结果显示,与A组相比,B组(P0.05)及C组(P0.01)肿瘤微环境中MDSC和M2型TAM比例下降,M1型TAM升高,mDC和CD8+T细胞比例增加。结论恩度联合负载mut1抗原多肽的DC-T细胞治疗可有效抑制肿瘤血管新生,明显减缓肿瘤生长,加剧肿瘤组织内的低氧,有效逆转了肿瘤微环境的免疫抑制,从而发挥了协同抗肿瘤效应。
[Abstract]:Objective the occurrence, invasion and metastasis of tumor are closely related to neovascularization of tumor. Therefore, inhibition of angiogenesis is a new hot spot to control tumor growth. Recombinant human endostatin (Endostar) is a drug that inhibits angiogenesis by inhibiting the growth of blood vessels. Dendritic cells (DCs) is the most functional antigen presenting cell (antigen-presenting cells, APCs), which regulates the inherent and inherent characteristics of the body, which is the most functional antigen presenting cell (antigen-presenting cells, APCs). The adaptive immune response plays an important role. Dendritic cells loaded with tumor antigen are one of the most widely studied biological treatments in recent years. In this experiment, the anti-tumor effect of C57BL/6 specific DC-T cells loaded with Mut1 antigen and the effect on the immune network within the tumor microloop were studied by the Lewis tumor bearing tumor model of the mice. One step is to clarify the anti tumor effect of the combination of degree of grace and cell immunotherapy, so as to provide experimental basis and theoretical basis for the combination of anti angiogenic molecular targeting therapy and cell immunotherapy. Method 1. Lewis lung cancer cells were cultured. Under the microscope, the state and quantity of cells were observed and the liquid and passages were changed in time, and the cell growth entered the logarithm. DC-T cells loaded with Mut1 antigen peptides were prepared by.2.: isolation and culture of mouse spleen T lymphocytes, isolation and culture of dendritic cells from mice, dendritic cells with Mut1 antigen peptide and T lymphocyte co culture to prepare Mut1 antigen specific DC-T cells in.3. construction of C57BL/6 mouse Lewis lung cancer subcutaneous implantation The tumor model was divided into groups: C57BL/6 mice were randomly divided into A group (PBS control group), group B (group DC-T) and group C (DC-T+ group). The treatment intervention was given seventh days after the tumor. A group was given the tail vein injection PBS solution, B and C groups were given tail vein infusion of the load MutL antipropeptide cells, and the group was given the tail vein injection Grace (recombinant human blood). The growth and tumor suppressor rate of.4. tumor were measured: the mass of the mice and the maximum diameter and short diameter of the transplanted tumor were measured every day after the tumor. The tumor volume was calculated and the growth curve was plotted. After the last intervention 24 hours, the mice were killed and the tumor tissue was dissected and the tumor suppressor rate was measured by the.5. immunohistochemical SP method. Tumor tissue microvessel density (MVD).6.Western blotting was used to detect the expression of VEGF-A and HIF-1 a in the tumor tissues..7. flow cytometry was used to detect the proportion of MDSC, TAM (M1/M2), mDC and CD8+T cells in tumor tissues. Results 1. compared with the A group, the mass and tumor volume of the mice increased slowly. The difference was statistically significant (P0.05), the tumor suppressor rate was 25%, and the tumor quality of group C was significantly smaller than that of group A, the difference was statistically significant (P0.01). The tumor inhibition rate was 52%.2.MVD count statistics showed that the MVD count decreased in B group and A group, and the difference was statistically significant (P0.05); C group and A group were significantly less than those in A group, and the difference was statistically significant. (P0.01).3.Western blotting results showed that the level of VEGF-A protein expression in B group (P0.05) and C group (P0.01) decreased, and the expression level of HIF-1 alpha protein increased by 4. flow cytometry. The proportion of CD8+T cells increased. Conclusion the combination of DC-T cell therapy with Mut1 antigen with the combination of grace and load can effectively inhibit tumor angiogenesis, significantly slow down the growth of tumor, aggravate the hypoxia in tumor tissue, effectively reverse the immunosuppression of tumor microenvironment, and thus play a synergistic antitumor effect.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2

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