【摘要】：[背景和目的] 惡性腫瘤是危害人類健康的主要疾病之一，傳統(tǒng)的手術(shù)、放、化療的療效又不盡人意，這種局限決定了生物治療必然要參與腫瘤的綜合治療，而腫瘤疫苗是腫瘤生物治療的重要方法。目前常用的腫瘤疫苗傳遞方式分別以細(xì)胞、蛋白質(zhì)、多肽、病毒載體為基礎(chǔ)。樹突細(xì)胞(Dendritic cells, DCs)是目前已知的抗原提呈功能最強(qiáng)的免疫細(xì)胞，將腫瘤相關(guān)抗原(Tumor-asscociated antigen, TAA)負(fù)載DC是目前常用的腫瘤免疫治療的方式之一。但目前發(fā)現(xiàn)的腫瘤相關(guān)抗原大多具有組織特異性，如AFP只針對肝癌等，以這些抗原來進(jìn)行免疫治療，只能對相應(yīng)的腫瘤起作用，限制了以這些抗原為靶標(biāo)的腫瘤疫苗的廣泛應(yīng)用。尋找腫瘤特異性的共有抗原是腫瘤免疫治療的關(guān)鍵問題。 人端粒酶逆轉(zhuǎn)錄酶，又名人端粒酶催化亞單位，為端粒酶活性的限速成分，是癌細(xì)胞永生化的必要途徑，在維持腫瘤繼續(xù)分裂、增殖和生存中發(fā)揮重要作用。國內(nèi)外許多研究表明，人端粒酶逆轉(zhuǎn)錄酶在85％以上的惡性腫瘤及其癌前病變中過度表達(dá)，其異常表達(dá)或激活是腫瘤形成的重要原因之一，新近研究報道人端粒酶逆轉(zhuǎn)錄酶的高表達(dá)也與腫瘤的不良預(yù)后、浸潤轉(zhuǎn)移均密切相關(guān)。我們過去的研究表明，帶有hTERT全長基因的腺病毒載體負(fù)載DC，以及帶有hTERT全長基因和隱性表位的顆粒疫苗，在體內(nèi)外均可以誘導(dǎo)產(chǎn)生hTERT特異性的細(xì)胞毒T淋巴細(xì)胞(CTLs)，對hTERT陽性的胃癌、結(jié)腸癌等多種癌細(xì)胞具有一定的殺傷效應(yīng)，而對表達(dá)hTERT陰性的淋巴細(xì)胞和DC不具有殺傷效應(yīng)。與hTERT全基因序列腺病毒負(fù)載DC疫苗相比，多肽疫苗沒有腺病毒載體的參與，不必考慮載體及其整合的安全性，也不必考慮全長蛋白中某些非優(yōu)勢表位或病理表位的毒副作用；而且，抗原肽長度僅8-15個左右的氨基酸序列，體外可以合成，臨床應(yīng)用前景更廣闊。但多肽疫苗也常有其不足之處：最大的問題在于一個多肽疫苗只能代表一個表位，分子量小，結(jié)構(gòu)單一，免疫原性較弱，在體內(nèi)容易降解。為解決這一弊端，1988年Tam實驗室提出了多抗原肽(Multiple antigen peptides,MAP)的設(shè)計方案，其聚賴氨酸核心構(gòu)成一種十分緊密有序的樹狀結(jié)構(gòu)，它在加強(qiáng)了抗原表位肽鏈結(jié)構(gòu)特異性的同時，還增大其分子質(zhì)量，不僅很好的模擬了天然表位的空間構(gòu)象，而且還不需要再耦聯(lián)載體蛋白就能誘導(dǎo)較強(qiáng)的免疫反應(yīng)；同時，其穩(wěn)定的二級結(jié)構(gòu)不易反轉(zhuǎn)，故降解速度減緩，延長抗原刺激時間，獲得更好的殺傷活性。MAP疫苗的設(shè)計方案不僅適用于B細(xì)胞表位，同樣也適用于T細(xì)胞表位。 基于以上分析，本實驗擬選取人端粒酶逆轉(zhuǎn)錄酶HLA-A2限制性CTL表位hTERT-540(ILAKFLHWL), hTERT-865(RLVDDFLLV) and hTERT-572Y(YLFFYRKSV)，構(gòu)建其四分支多抗原肽，研究其在體外體內(nèi)的抗腫瘤活性，并與攜帶hTERT全長基因腺病毒載體疫苗、單肽疫苗誘導(dǎo)的CTL活性進(jìn)行對比，為hTERT的MAP疫苗抗腫瘤效應(yīng)的臨床應(yīng)用提供理論依據(jù)。 [方法] 1、選取三條人端粒酶逆轉(zhuǎn)錄酶CTL表位［hTERT-540(ILAKFLHWL), hTERT-865(RLVDDFLLV) and hTERT-572Y (YLFFYRKSV)］設(shè)計其MAP結(jié)構(gòu)，采用標(biāo)準(zhǔn)固相肽合成法(SPSS)合成其四分支肽；反相高壓液相色譜儀(RP-HPLC) 分析其純度；液相色譜/質(zhì)譜聯(lián)用(LC/MS)檢測多肽分子量。陰性肽選用人 HIV病毒HLA-A2限制性表位(ILLEP VHGV)。 2、按文獻(xiàn)所述分離培養(yǎng)人外周血單個核細(xì)胞(PBMC)來源的樹突細(xì)胞。采用光學(xué)顯微鏡觀察其形態(tài)，并采用流式細(xì)胞術(shù)鑒定其細(xì)胞表型。然后將攜帶hTERT全長序列的腺病毒載體、人端粒酶逆轉(zhuǎn)錄酶MAP肽、及其對應(yīng)單肽分別負(fù)載DC后，誘導(dǎo)產(chǎn)生人端粒酶逆轉(zhuǎn)錄酶特異性CTL。采用標(biāo)準(zhǔn)51Cr釋放實驗檢測人端粒酶逆轉(zhuǎn)錄酶特異性CTL對不同腫瘤靶細(xì)胞[SW480結(jié)腸癌細(xì)胞(hTERT~+, HLA-A2~+)、KATO-Ⅲ胃癌細(xì)胞(hTERT~+, HLA-A2~+)、U2OS骨肉瘤細(xì)胞(hTERT-, HLA-A2~+),轉(zhuǎn)染了人端粒酶逆轉(zhuǎn)錄酶cDNA的U2OS/hTERT骨肉瘤細(xì)胞(hTERT~+, HLA-A2~+), MCF-7乳腺癌細(xì)胞(hTERT~+, HLA-A2~+), HepG2肝癌細(xì)胞(hTERT~+, HLA-A2-),轉(zhuǎn)染了HLA-A2cDNA的HepG2/HLA-A2肝癌細(xì)胞(hTERT~+, HLA-A2~+)的免疫殺傷活性；采取同樣方法檢測上述人端粒酶逆轉(zhuǎn)錄酶特異性CTL對低表達(dá)人端粒酶逆轉(zhuǎn)錄酶的自體淋巴細(xì)胞和樹突細(xì)胞有無殺傷作用以研究其毒副作用；采用ELISPOT試驗檢測產(chǎn)IFN-γ效應(yīng)細(xì)胞的數(shù)量。 3、按文獻(xiàn)所述分離培養(yǎng)C57BL/6-Tg(HLA-A2~+)小鼠骨髓來源的樹突細(xì)胞(mDC)， 采用光學(xué)顯微鏡觀察其形態(tài)，采用流式細(xì)胞術(shù)鑒定其細(xì)胞表型。然后將攜帶hTERT全長序列的腺病毒載體、人端粒酶逆轉(zhuǎn)錄酶MAP肽、及其對應(yīng)單肽分別負(fù)載mDC后，免疫小鼠，每周一次，共三次。最后一次免疫7天后取免疫小鼠脾淋巴細(xì)胞作為效應(yīng)細(xì)胞，51Cr釋放實驗檢測人端粒酶逆轉(zhuǎn)錄酶特異性CTL對上述的腫瘤靶細(xì)胞以及自體淋巴細(xì)胞和樹突狀細(xì)胞有無殺傷效應(yīng)；采用ELISPOT試驗檢測產(chǎn)IFN-γ效應(yīng)細(xì)胞的數(shù)量。 4、實驗數(shù)據(jù)采用SPSS17.0統(tǒng)計軟件進(jìn)行t檢驗，當(dāng)P㩳0.05時認(rèn)為差異有統(tǒng)計學(xué)意義。 ［結(jié)果］ 1、通過反相高壓液相色譜儀(RP-HPLC)檢測合成的多抗原肽及對應(yīng)單肽的純度均在95%以上，達(dá)到國際多肽實驗標(biāo)準(zhǔn)。液相色譜/質(zhì)譜聯(lián)用(LC/MS)檢測合成的多抗原肽及對應(yīng)單肽的分子量，結(jié)果表明所得肽的分子量理論值與實測值之間無明顯差異，為我們所需的目的多肽。 2、體外(In vitro)和離體(Ex vivo)實驗均提示，攜帶全長hTERT序列的腺病毒載體、人端粒酶逆轉(zhuǎn)錄酶特異性CTL對于人端粒酶逆轉(zhuǎn)錄酶陽性且HLA-A2陽性的SW480、MCF-7、KATO-Ⅲ細(xì)胞具有明顯的殺傷作用，且攜帶全長hTERT序列的腺病毒載體誘導(dǎo)的殺傷效應(yīng)最強(qiáng)；人端粒酶逆轉(zhuǎn)錄酶MAP所誘導(dǎo)的殺傷效應(yīng)明顯高于其對應(yīng)單肽誘導(dǎo)的殺傷效應(yīng)；但對HLA-A2陽性但人端粒酶逆轉(zhuǎn)錄酶陰性的U2OS細(xì)胞或人端粒酶逆轉(zhuǎn)錄酶陽性但HLA-A2陰性的HepG2細(xì)胞均無殺傷效應(yīng)，對人端粒酶逆轉(zhuǎn)錄酶陽性且HLA-A2.1匹配的U2OS/hTERT細(xì)胞和HepG2/HLA-A2.1細(xì)胞，提示該CTL反應(yīng)具有人端粒酶逆轉(zhuǎn)錄酶特異性及HLA-A2限制性。同時本實驗還發(fā)現(xiàn)，無論是人端粒酶逆轉(zhuǎn)錄酶MAP肽誘導(dǎo)的CTL，還是其對應(yīng)單肽誘導(dǎo)的效應(yīng)細(xì)胞對低表達(dá)人端粒酶逆轉(zhuǎn)錄酶的自體淋巴細(xì)胞和DC都不具有殺傷效應(yīng)，提示人端粒酶逆轉(zhuǎn)錄酶MAP肽疫苗的安全性。通過ELISPOT試驗檢測產(chǎn)IFN-γ效應(yīng)細(xì)胞的數(shù)量，結(jié)果顯示攜帶全長hTERT序列的腺病毒載體可以誘導(dǎo)效應(yīng)細(xì)胞釋放最多IFN-γ；人端粒酶逆轉(zhuǎn)錄酶MAP肽較之其對應(yīng)單肽可以誘導(dǎo)更多的效應(yīng)細(xì)胞產(chǎn)生IFN-γ。但在去除CD4＋T淋巴細(xì)胞后，攜帶全長hTERT序列的腺病毒載體、人端粒酶逆轉(zhuǎn)錄酶MAP肽及其對應(yīng)單肽誘導(dǎo)效應(yīng)細(xì)胞分泌IFN-γ的水平均明顯下降，，提示CD4+T淋巴細(xì)胞在CD8+T淋巴細(xì)胞的免疫應(yīng)答及應(yīng)答維持中發(fā)揮重要作用。 ［結(jié)論］ DC負(fù)載的HLA-A2限制性人端粒酶逆轉(zhuǎn)錄酶CTL表位MAP疫苗相較于其單肽能在體外體內(nèi)誘導(dǎo)更強(qiáng)的抗腫瘤免疫效應(yīng)。這種人端粒酶逆轉(zhuǎn)錄酶MAP疫苗具有高效、安全的特點，為人端粒酶逆轉(zhuǎn)錄酶MAP肽疫苗的臨床應(yīng)用提供了實驗依據(jù)。
[Abstract]:[background and purpose]
Malignant tumor is one of the main diseases that endanger human health. The efficacy of traditional surgery, radiotherapy and chemotherapy is unsatisfactory. This limitation determines that biotherapy must participate in the comprehensive treatment of tumor. Cancer vaccine is an important method of tumor biotherapy. Currently, the commonly used delivery methods of tumor vaccine are cell, protein, polypeptide. Dendritic cells (DCs) are the most potent antigen-presenting immune cells. Tumor-associated antigen (TAA) loaded with DCs is one of the most commonly used methods for tumor immunotherapy. Immunotherapy with these antigens can only work on the corresponding tumors, which limits the wide application of tumor vaccines targeting these antigens.
Human telomerase reverse transcriptase, also known as human telomerase catalytic subunit, is the rate-limiting component of telomerase activity and is an essential pathway for immortalization of cancer cells. It plays an important role in maintaining the continued division, proliferation and survival of tumors. Overexpression, abnormal expression or activation of human telomerase reverse transcriptase is one of the important causes of tumor formation. Recent studies have reported that high expression of human telomerase reverse transcriptase is also closely related to the poor prognosis, invasion and metastasis of tumors. Epitope-specific granular vaccines can induce hTERT-specific cytotoxic T lymphocytes (CTLs) in vitro and in vivo, and have a certain killing effect on hTERT-positive gastric cancer, colon cancer and other cancer cells, but have no killing effect on hTERT-negative lymphocytes and DC. Polypeptide vaccines have no adenovirus vectors, no need to consider the safety of vectors and their integration, and no need to consider the toxic and side effects of some non-dominant epitopes or pathological epitopes in full-length proteins. Moreover, antigen peptides with only 8-15 amino acid sequences can be synthesized in vitro and have a broader clinical application prospect. The biggest problem is that a polypeptide vaccine can only represent one epitope, with small molecular weight, single structure, weak immunogenicity and easy degradation in vivo. To solve this problem, Tam Laboratory put forward a design scheme of multiple antigen peptides (MAP) in 1988. Its polylysine core constitutes one kind of 10. Tightly ordered dendritic structures not only enhance the structural specificity of antigen epitope peptides, but also increase their molecular weight. They not only simulate the spatial conformation of natural epitopes, but also induce strong immune responses without recombinant carrier proteins. At the same time, their stable secondary structure is not easy to reverse, so the degradation rate is fast. The design of MAP vaccine is not only suitable for B cell epitope, but also for T cell epitope.
Based on the above analysis, we intend to construct four-branched polyantigenic peptides of human telomerase reverse transcriptase HLA-A2 restriction CTL epitopes hTERT-540 (ILAKFLHWL), hTERT-865 (RLVDDFLLV) and hTERT-572Y (YLFFYRKSV) to study their antitumor activities in vitro and in vivo, and to induce them with adenovirus vector vaccine carrying full-length hTERT gene and single peptide vaccine. The comparison of CTL activity provides a theoretical basis for the clinical application of hTERT MAP vaccine.
[method]
1. Three human telomerase reverse transcriptase CTL epitopes (hTERT-540 (ILAKFLHWL), hTERT-865 (RLVDDFLLV) and hTERT-572Y (YLFFYRKSV) were selected to design their MAP structures, and their tetrabranched peptides were synthesized by standard solid-phase peptide synthesis (SPSS); and their tetrabranched peptides were synthesized by RP-HPLC.
The purity of the peptide was analyzed by liquid chromatography / mass spectrometry (LC/MS).
HIV virus HLA-A2 restricted epitope (ILLEP VHGV).
2. Human peripheral blood mononuclear cells (PBMC) derived dendritic cells were isolated and cultured according to the literature. The morphology of PBMC was observed by optical microscope and its phenotype was identified by flow cytometry. The adenovirus vector carrying full-length sequence of hTERT, human telomerase reverse transcriptase MAP peptide and its corresponding monopeptide were loaded with DC respectively to induce the production of dendritic cells. Human telomerase reverse transcriptase-specific CTL. Human telomerase reverse transcriptase-specific CTL was transfected into human telomerase reverse transcriptase cDNA U2OS/hTER by standard 51Cr release assay in different tumor target cells [SW480 colon cancer cells (hTERT~+, HLA-A2~+), KATO-III gastric cancer cells (hTERT~+, HLA-A2~+), U2OS osteosarcoma cells (hTERT-, HLA-A2~+). Immunocytotoxicity of HPG2/HLA-A2 hepatoma cells (hTERT~+, HLA-A2~+), MCF-7 breast cancer cells (hTERT~+, HLA-A2~+), HepG2 hepatoma cells (hTERT~+, HLA-A2 -) transfected with HLA-A2 cDNA was detected by the same method. The number of IFN-gamma effector cells was detected by ELISPOT assay.
3, according to the literature, the bone marrow derived dendritic cells (mDC) of C57BL/6-Tg (HLA-A2~+) mice were isolated and cultured.
Then the adenovirus vector carrying the full-length sequence of hTERT, human telomerase reverse transcriptase MAP peptide and its corresponding single peptide were loaded with mDC respectively, and the mice were immunized once a week for three times. The spleen lymphocytes of the immunized mice were obtained after the last immunization for seven days. The cytotoxicity of human telomerase reverse transcriptase-specific CTL to tumor target cells, autologous lymphocytes and dendritic cells was detected by 51Cr release assay, and the number of IFN-gamma effector cells was detected by ELISPOT assay.
4, the experimental data were analyzed by SPSS17.0 statistical software for t test. When P? 0.05, the difference was statistically significant.
[results]
1. The purity of the polyantigenic peptides and the corresponding single peptides were all above 95% by RP-HPLC. The molecular weight of the polyantigenic peptides and the corresponding single peptides were determined by liquid chromatography/mass spectrometry (LC/MS). The results showed that there was no obvious difference between the theoretical value and the measured value of the molecular weight of the peptides. Differences are the peptides we need.
2. In vitro and Ex vivo experiments showed that adenovirus vector carrying full-length hTERT sequence and human telomerase reverse transcriptase-specific CTL had significant killing effect on SW480, MCF-7 and KATO-III cells with positive human telomerase reverse transcriptase and HLA-A2, and the adenovirus vector carrying full-length hTERT sequence induced killing effect. The killing effect induced by human telomerase reverse transcriptase MAP was significantly higher than that induced by its corresponding single peptide, but it had no killing effect on HLA-A2 positive U2OS cells or HLA-A2 negative HepG2 cells. LA-A2.1-matched U2OS/hTERT cells and HepG2/HLA-A2.1 cells suggested that the CTL reaction was human telomerase reverse transcriptase-specific and HLA-A2-restrictive. It was also found that either the CTL induced by human telomerase reverse transcriptase MAP peptide or the effector cells induced by its corresponding single peptide could induce autologous lymphoma of low-expression human telomerase reverse transcriptase. The number of IFN-gamma effector cells was detected by ELISPOT assay. The results showed that adenovirus vector carrying full-length hTERT sequence could induce effector cells to release the most IFN-gamma. Human telomerase reverse transcriptase MAP peptide was more safe than its corresponding single peptide. More effector cells could be induced to produce IFN-gamma. However, after removal of CD4+T lymphocytes, adenovirus vector carrying full-length hTERT sequence, the levels of IFN-gamma secreted by human telomerase reverse transcriptase MAP peptide and its corresponding monopeptide-induced effector cells were significantly decreased, suggesting that CD4+T lymphocytes maintained immune response and response in CD8+T lymphocytes. China plays an important role.
[Conclusion]
DC-loaded HLA-A2 restriction human telomerase reverse transcriptase CTL epitope MAP vaccine can induce stronger anti-tumor immune effect in vitro than its single peptide. This human telomerase reverse transcriptase MAP vaccine has the characteristics of high efficiency and safety, which provides experimental basis for clinical application of human telomerase reverse transcriptase MAP peptide vaccine.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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相關(guān)期刊論文 前1條

1 王kH;誘導(dǎo)免疫應(yīng)答的腫瘤抗原[J];生命科學(xué);2002年01期

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