本文選題：CD133 + 表位　； 參考：《河北大學》2017年碩士論文

【摘要】：白血病是青少年常見的惡性腫瘤之一,由于化療、骨髓移植和嵌合抗原受體T細胞(CAR T)治療技術的逐漸提高,使得白血病的5年存活率達62%以上。然而,白血病的復發(fā)和難治療性的問題仍然比較嚴峻。雖然臨床試驗已經有藥物用于靶向治療白血病,但是耐藥性和毒副作用等問題比較顯著。由于腫瘤特異性T細胞,特別是CD8+T細胞(CTL)在殺傷和清除腫瘤中發(fā)揮重要作用,因此研究新型特異性高、安全性好的腫瘤免疫治療性疫苗用于活化特異性CTL,是應對白血病的有效手段之一。腫瘤干細胞因為具備自我更新、無限增殖和分化形成腫瘤的能力,并且對化療、放療具有抗性,被認為是腫瘤復發(fā)的根源所在。多種腫瘤中都發(fā)現了CD133~+腫瘤干細胞,提示CD133抗原可以作為CTL識別的靶點,這也為設計新型白血病治療性疫苗提供了線索。熱休克蛋白gp96作為分子伴侶不僅可以激活T細胞免疫,同時也可以激活先天免疫。本研究首先利用昆蟲細胞表達系統,表達有免疫學功能的重組gp96蛋白。通過表位預測,發(fā)現CD133含有6個潛在H2-Kd限制性的表位,分別是CD133272-281(QNMSSSLKSL)、CD133419-428(RYLNQELPKL)、CD133452-461(FFFLGLLCGV)、CD133601-609(NLNVNIDSI)、CD133702-710(TLRQSVWTL)和CD133760-769(HYLHWVFYAI)。進一步將重組gp96作為T細胞疫苗佐劑,通過小鼠免疫實驗鑒定出3個可活化特異性的CTL表位,分別是CD133419-428、CD133702-710和CD133760-769。在此基礎上,將這三個表位聯合gp96佐劑制備新型表位疫苗,在小鼠CD133~+淋巴細胞性白血病的模型中,CD133表位疫苗引發(fā)抗腫瘤特異性T細胞免疫應答。在預防模型中,免疫表位疫苗后小鼠腫瘤體積和重量與對照組相比都下降了43%(P0.05);ELISPOT實驗證明表位疫苗免疫后CD133419-428、CD133702-710和CD133760-769表位特異的T細胞比對照組分別增加了4.6、0.6和1.2倍;分離免疫后小鼠的脾臟T細胞進行細胞毒性實驗,發(fā)現當效應細胞與靶細胞比為20:1時,表位疫苗組對靶細胞的特異性殺傷作用明顯地高于對照組(P0.05)。在治療模型中,免疫表位疫苗后小鼠腫瘤體積和重量與對照相比分別下降了31%(P0.05)和30%(P0.05);分離免疫后小鼠的脾臟T細胞進行細胞毒性實驗,發(fā)現當效應細胞與靶細胞比為20:1時,表位疫苗組對靶細胞的特異性殺傷作用極顯著地高于對照組(P0.01)。最后,在荷瘤小鼠體內轉輸CD133表位疫苗激活的特異T細胞,能顯著地抑制腫瘤生長。綜上所述,本研究以小鼠淋巴細胞性白血病為模型,設計了以gp96為佐劑的、新型的抗CD133~+腫瘤的治療性表位疫苗,通過小鼠實驗驗證該疫苗能夠活化CD133特異性CTL應答,顯著抑制小鼠淋巴瘤的生長,這為進一步研發(fā)靶向CD133~+的白血病等腫瘤治療性疫苗提供了依據。
[Abstract]:Leukemia is one of the most common malignant tumors in adolescents. Because of chemotherapy, bone marrow transplantation and treatment of chimeric antigen receptor T cells (car T), the 5-year survival rate of leukemia is over 62%. However, the recurrence of leukemia and difficult to treat the problem is still relatively serious. Although drugs have been used to target leukemia in clinical trials, drug resistance and side effects are significant. Because tumor specific T cells, especially CD8 T cells (CTL), play an important role in killing and clearing tumors, new types of tumor specific T cells are studied with high specificity. Tumor immunotherapy vaccine with good safety is one of the effective methods for activating specific CTLs. Tumor stem cells have the ability of self-renewal, infinite proliferation and differentiation to form tumors, and are resistant to chemotherapy and radiotherapy, which is considered to be the root cause of tumor recurrence. CD133- tumor stem cells have been found in many kinds of tumors, suggesting that CD133 antigen can be used as a target for CTL recognition, which provides clues for the design of a new therapeutic vaccine for leukemia. Heat shock protein (gp96), as a molecular chaperone, not only activates T cell immunity, but also activates innate immunity. In this study, the recombinant gp96 protein with immunological function was expressed by insect cell expression system. By epitope prediction, it was found that CD133 contained six potential H2-Kd restricted epitopes, CD133272-281 (QNMSSSLKSL), CD133419-428 (RYLNQELPKL) CD133452-461 (FFLGLLCGV) CD133601-609 (NLNVNIDSI) CD133702-710 (TLRQSVWTL) and CD133760-769 (HYLHWVFYAI). The recombinant gp96 was further used as the adjuvant of T cell vaccine. Three specific CTL epitopes, CD133419-428, CD133702-710 and CD133760-769, were identified by immunological assay. On this basis, the three epitopes combined with gp96 adjuvant were used to prepare the novel epitope vaccine, and CD133 epitope vaccine induced anti-tumor specific T cell immune response in mouse CD133- lymphocytic leukemia model. In the preventive model, the tumor volume and weight of mice after immunization with epitope vaccine decreased by 43% (P0.05) compared with the control group (P0.05). The results of Elisa showed that the epitope specific T cells of CD133419-428 and CD133702-710 and CD133760-769 increased 4.60.6 and 1.2 times, respectively, compared with the control group. When the ratio of effector cells to target cells was 20:1, the specific killing effect of epitope vaccine group on target cells was significantly higher than that of control group (P0.05). In the treatment model, the tumor volume and weight of mice decreased by 31% (P0.05) and 30% (P0.05), respectively, compared with the control group, and the cytotoxicity test of spleen T cells of the immunized mice showed that when the ratio of effector cells to target cells was 20:1, the tumor size and weight of mice decreased by 31% (P0.05) and 30% (P0.05) respectively after immunization, and the ratio of effector cells to target cells was 20:1. The specific killing effect of epitope vaccine group on target cells was significantly higher than that of control group (P0.01). Finally, transfusions of specific T cells activated by CD133 epitope vaccine in tumor-bearing mice significantly inhibited tumor growth. To sum up, a novel therapeutic epitope vaccine against CD133~ tumor was designed using gp96 as adjuvant in murine lymphoblastic leukemia model. The results of mouse experiments showed that the vaccine could activate CD133-specific CTL response. The growth of murine lymphoma was significantly inhibited, which provided a basis for the further development of tumor therapeutic vaccines targeting CD133- to leukemia.
【學位授予單位】：河北大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.7

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本文編號：2099779