本文關(guān)鍵詞： 樹突狀細胞 細胞毒性T淋巴細胞 K-ras 肺癌　出處：《鄭州大學》2017年博士論文　論文類型：學位論文

【摘要】：目的:肺癌是當今世界上最常見的惡性腫瘤之一,也是當前我國腫瘤死亡的主要原因之一。由于早期肺癌患者往往缺乏明顯的臨床癥狀,導致75%的患者就診時已失去根治性手術(shù)的機會,而姑息性手術(shù)或常規(guī)放化療僅能夠部分改善癥狀,無法獲得良好的緩解率,從而使得肺癌患者的中位生存期僅能達到12~18個月。除此之外,生物治療也已經(jīng)在臨床治療肺癌方面得到了廣泛的應用,主要包括基因治療及免疫治療。而免疫治療主要包括有細胞因子治療、特異性主動免疫治療和過繼性細胞免疫治療等。在人體內(nèi)存在許多種抗原提呈細胞(Antigen presenting cell,APC,其中功能最為強大的是樹突狀細胞(Dendritic cells,DC),因此人們也越來越注意到樹突狀細胞在肺癌治療中的作用。近年來,大量研究表明K-ras基因突變是非小細胞肺癌發(fā)展過程中的一個重要的負性預后因素,而K-ras基因已成為肺癌生物治療的一個重要靶位。本研究擬通過在體和體外實驗,探討負載不同抗原的DC誘導的腫瘤特異性CTL對肺癌細胞的殺傷效應及其對荷瘤裸鼠的抑制作用。方法:聯(lián)合應用粒細胞-巨噬細胞集落刺激因子(rh GM-CSF)和白細胞介素4(IL-4)誘導外周血樹突狀細胞(DC),分別使用表達K-ras突變體的肺癌細胞抗原、單純K-ras突變體抗原表位肽和K-ras突變體抗原表位肽陽離子納米顆粒致敏DC,進而通過誘導刺激T淋巴細胞得到特異性的CTL。流式細胞儀測定DC細胞表面標志,MTT法檢測CTL細胞在體外對腫瘤細胞的殺傷作用,ELISA試劑盒檢測IL-12和IFN-γ表達水平。分別使用肺癌A549、NCH-446細胞系建立荷瘤裸鼠模型評價CTL體內(nèi)抗腫瘤活性。結(jié)果:在體外實驗中,與單純K-ras突變體多肽相比,K-ras突變體表位肽陽離子納米顆粒在低濃度時即可被DC有效提呈(P0.05);與負載單純K-ras突變體多肽和K-ras突變體表位肽陽離子納米顆粒組相比,負載全瘤抗原組DC誘導產(chǎn)生的CTL對A549(K-ras+)和NCH-446(K-ras-)均有明顯殺傷活性(P0.05);而負載單純K-ras突變體多肽、K-ras突變體表位肽陽離子納米顆粒的DC誘導產(chǎn)生的CTL對A549(K-ras+)有特異性殺傷作用,而對NCH-446(K-ras-)細胞無殺傷作用(P0.05)。體內(nèi)實驗結(jié)果顯示,負載全瘤抗原的DC誘導產(chǎn)生的CTL對表達K-ras陽性(A549)的肺癌細胞具有較好的抑制作用,而對K-ras表達陰性(NCH-446)的肺癌細胞抑制作用與對照組相比無明顯差異(P0.05)。結(jié)論:低濃度的K-ras突變體表位肽陽離子納米顆粒作用后,短時間內(nèi)即可被DC有效提呈,且其誘導產(chǎn)生的CTL對表達K-ras突變體的肺癌細胞有特異性的殺傷作用;而負載腫瘤抗原的DC誘導CTL能夠顯著抑制腫瘤的生長速度進而提高荷瘤裸鼠的生存時間,這一結(jié)果為以后的研究奠定了基礎(chǔ),進而可以用于臨床。
[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- 緯 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.
【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R734.2

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