本文關(guān)鍵詞： 樹(shù)突狀細(xì)胞 細(xì)胞毒性T淋巴細(xì)胞 K-ras 肺癌　出處：《鄭州大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：目的:肺癌是當(dāng)今世界上最常見(jiàn)的惡性腫瘤之一,也是當(dāng)前我國(guó)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(dǎo)致75%的患者就診時(shí)已失去根治性手術(shù)的機(jī)會(huì),而姑息性手術(shù)或常規(guī)放化療僅能夠部分改善癥狀,無(wú)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(dá)到12~18個(gè)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(yīng)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(xì)胞因子治療、特異性主動(dòng)免疫治療和過(guò)繼性細(xì)胞免疫治療等。在人體內(nèi)存在許多種抗原提呈細(xì)胞(Antigen presenting cell,APC,其中功能最為強(qiáng)大的是樹(shù)突狀細(xì)胞(Dendritic cells,DC),因此人們也越來(lái)越注意到樹(shù)突狀細(xì)胞在肺癌治療中的作用。近年來(lái),大量研究表明K-ras基因突變是非小細(xì)胞肺癌發(fā)展過(guò)程中的一個(gè)重要的負(fù)性預(yù)后因素,而K-ras基因已成為肺癌生物治療的一個(gè)重要靶位。本研究擬通過(guò)在體和體外實(shí)驗(yàn),探討負(fù)載不同抗原的DC誘導(dǎo)的腫瘤特異性CTL對(duì)肺癌細(xì)胞的殺傷效應(yīng)及其對(duì)荷瘤裸鼠的抑制作用。方法:聯(lián)合應(yīng)用粒細(xì)胞-巨噬細(xì)胞集落刺激因子(rh GM-CSF)和白細(xì)胞介素4(IL-4)誘導(dǎo)外周血樹(shù)突狀細(xì)胞(DC),分別使用表達(dá)K-ras突變體的肺癌細(xì)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(yáng)離子納米顆粒致敏DC,進(jìn)而通過(guò)誘導(dǎo)刺激T淋巴細(xì)胞得到特異性的CTL。流式細(xì)胞儀測(cè)定DC細(xì)胞表面標(biāo)志,MTT法檢測(cè)CTL細(xì)胞在體外對(duì)腫瘤細(xì)胞的殺傷作用,ELISA試劑盒檢測(cè)IL-12和IFN-γ表達(dá)水平。分別使用肺癌A549、NCH-446細(xì)胞系建立荷瘤裸鼠模型評(píng)價(jià)CTL體內(nèi)抗腫瘤活性。結(jié)果:在體外實(shí)驗(yàn)中,與單純K-ras突變體多肽相比,K-ras突變體表位肽陽(yáng)離子納米顆粒在低濃度時(shí)即可被DC有效提呈(P0.05);與負(fù)載單純K-ras突變體多肽和K-ras突變體表位肽陽(yáng)離子納米顆粒組相比,負(fù)載全瘤抗原組DC誘導(dǎo)產(chǎn)生的CTL對(duì)A549(K-ras+)和NCH-446(K-ras-)均有明顯殺傷活性(P0.05);而負(fù)載單純K-ras突變體多肽、K-ras突變體表位肽陽(yáng)離子納米顆粒的DC誘導(dǎo)產(chǎn)生的CTL對(duì)A549(K-ras+)有特異性殺傷作用,而對(duì)NCH-446(K-ras-)細(xì)胞無(wú)殺傷作用(P0.05)。體內(nèi)實(shí)驗(yàn)結(jié)果顯示,負(fù)載全瘤抗原的DC誘導(dǎo)產(chǎn)生的CTL對(duì)表達(dá)K-ras陽(yáng)性(A549)的肺癌細(xì)胞具有較好的抑制作用,而對(duì)K-ras表達(dá)陰性(NCH-446)的肺癌細(xì)胞抑制作用與對(duì)照組相比無(wú)明顯差異(P0.05)。結(jié)論:低濃度的K-ras突變體表位肽陽(yáng)離子納米顆粒作用后,短時(shí)間內(nèi)即可被DC有效提呈,且其誘導(dǎo)產(chǎn)生的CTL對(duì)表達(dá)K-ras突變體的肺癌細(xì)胞有特異性的殺傷作用;而負(fù)載腫瘤抗原的DC誘導(dǎo)CTL能夠顯著抑制腫瘤的生長(zhǎng)速度進(jìn)而提高荷瘤裸鼠的生存時(shí)間,這一結(jié)果為以后的研究奠定了基礎(chǔ),進(jìn)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(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2

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