负载K-ras抗原的树突状细胞诱导特异性细胞毒性T淋巴细胞（CTLs）对肺癌细胞的杀伤作用

发布时间：2018-01-21 02:05

本文关键词： 树突状细胞细胞毒性T淋巴细胞 K-ras 肺癌　出处：《郑州大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:肺癌是当今世界上最常见的恶性肿瘤之一,也是当前我国肿瘤死亡的主要原因之一。由于早期肺癌患者往往缺乏明显的临床症状,导致75%的患者就诊时已失去根治性手术的机会,而姑息性手术或常规放化疗仅能够部分改善症状,无法获得良好的缓解率,从而使得肺癌患者的中位生存期仅能达到12~18个月。除此之外,生物治疗也已经在临床治疗肺癌方面得到了广泛的应用,主要包括基因治疗及免疫治疗。而免疫治疗主要包括有细胞因子治疗、特异性主动免疫治疗和过继性细胞免疫治疗等。在人体内存在许多种抗原提呈细胞(Antigen presenting cell,APC,其中功能最为强大的是树突状细胞(Dendritic cells,DC),因此人们也越来越注意到树突状细胞在肺癌治疗中的作用。近年来,大量研究表明K-ras基因突变是非小细胞肺癌发展过程中的一个重要的负性预后因素,而K-ras基因已成为肺癌生物治疗的一个重要靶位。本研究拟通过在体和体外实验,探讨负载不同抗原的DC诱导的肿瘤特异性CTL对肺癌细胞的杀伤效应及其对荷瘤裸鼠的抑制作用。方法:联合应用粒细胞-巨噬细胞集落刺激因子(rh GM-CSF)和白细胞介素4(IL-4)诱导外周血树突状细胞(DC),分别使用表达K-ras突变体的肺癌细胞抗原、单纯K-ras突变体抗原表位肽和K-ras突变体抗原表位肽阳离子纳米颗粒致敏DC,进而通过诱导刺激T淋巴细胞得到特异性的CTL。流式细胞仪测定DC细胞表面标志,MTT法检测CTL细胞在体外对肿瘤细胞的杀伤作用,ELISA试剂盒检测IL-12和IFN-γ表达水平。分别使用肺癌A549、NCH-446细胞系建立荷瘤裸鼠模型评价CTL体内抗肿瘤活性。结果:在体外实验中,与单纯K-ras突变体多肽相比,K-ras突变体表位肽阳离子纳米颗粒在低浓度时即可被DC有效提呈(P0.05);与负载单纯K-ras突变体多肽和K-ras突变体表位肽阳离子纳米颗粒组相比,负载全瘤抗原组DC诱导产生的CTL对A549(K-ras+)和NCH-446(K-ras-)均有明显杀伤活性(P0.05);而负载单纯K-ras突变体多肽、K-ras突变体表位肽阳离子纳米颗粒的DC诱导产生的CTL对A549(K-ras+)有特异性杀伤作用,而对NCH-446(K-ras-)细胞无杀伤作用(P0.05)。体内实验结果显示,负载全瘤抗原的DC诱导产生的CTL对表达K-ras阳性(A549)的肺癌细胞具有较好的抑制作用,而对K-ras表达阴性(NCH-446)的肺癌细胞抑制作用与对照组相比无明显差异(P0.05)。结论:低浓度的K-ras突变体表位肽阳离子纳米颗粒作用后,短时间内即可被DC有效提呈,且其诱导产生的CTL对表达K-ras突变体的肺癌细胞有特异性的杀伤作用;而负载肿瘤抗原的DC诱导CTL能够显著抑制肿瘤的生长速度进而提高荷瘤裸鼠的生存时间,这一结果为以后的研究奠定了基础,进而可以用于临床。
[Abstract]:Objective: lung cancer is one of the most common malignant tumors in the world and one of the main causes of cancer death in China. As a result, 75% of the patients had lost the opportunity of radical surgery, while palliative surgery or routine radiotherapy and chemotherapy could only partially improve the symptoms and could not obtain a good remission rate. So that the median survival time of lung cancer patients can only reach 12 to 18 months. In addition, biological therapy has been widely used in the clinical treatment of lung cancer. Immunotherapy includes gene therapy and immunotherapy, and immunotherapy mainly includes cytokine therapy. Specific active immunotherapy and adoptive cellular immunotherapy. There are many kinds of antigen-presenting cells (APCs) in human body. Among them, dendritic cells are the most powerful, so people pay more and more attention to the role of dendritic cells in the treatment of lung cancer in recent years. A large number of studies have shown that K-ras gene mutation is an important negative prognostic factor in the development of non-small cell lung cancer. K-ras gene has become an important target of biological therapy for lung cancer. This study is intended to be carried out in vivo and in vitro. To investigate the cytotoxicity of tumor-specific CTL induced by DC loaded with different antigens on lung cancer cells and its inhibitory effect on tumor-bearing nude mice. Methods: granulocyte-macrophage colony stimulating factor (GSCF) was used in combination. Rh GM-CSF and IL-4) induced DC). Lung cancer cell antigen expressing K-ras mutant, simple K-ras mutant antigen epitope peptide and K-ras mutant antigen epitope peptide cationic nanoparticles were used to sensitize DC. The specific CTL. flow cytometry was used to detect the cytotoxicity of CTL cells to tumor cells in vitro. The expression levels of IL-12 and IFN- 纬 were detected by ELISA kit. A549 was used respectively. NCH-446 cell line was used to establish tumor-bearing nude mice model to evaluate the anti-tumor activity of CTL in vivo. Results: compared with K-ras mutant polypeptide in vitro. K-ras mutant peptide cationic nanoparticles can be effectively presented by DC at low concentration. Compared with the group loaded with pure K-ras mutant peptide and K-ras mutant peptide cationic nanoparticles. CTL induced by dendritic cells loaded with whole tumor antigen had significant cytotoxicity to both A549 K-ras and NCH-446 K-ras-containing cells. But the CTL produced by DC loaded with pure K-ras mutant peptide of K-ras mutant peptide cationic nanoparticles had a specific killing effect on A549-K-ras. However, NCH-446 K- ras-cell had no killing effect on NCH-446 K-ras-cell line (P0.05N). CTL induced by DC loaded with whole tumor antigen could inhibit the expression of K-ras positive A549) in lung cancer cells. The inhibitory effect of K-ras negative expression of NCH-446) on lung cancer cells was not significantly different from that of the control group (P0.05). Conclusion: low concentration of K ras mutant peptide cationic nanoparticles can be used. The CTL induced by DC could be effectively presented in a short time, and it had a specific killing effect on lung cancer cells expressing K-ras mutant. CTL induced by DC loaded with tumor antigen could significantly inhibit the growth rate of tumor and increase the survival time of tumor-bearing nude mice. The results laid a foundation for future research and could be used in clinical practice.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

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