【作者】 曹现祥；

【导师】 赵耀瑞； 徐勇； 张志宏；

【作者基本信息】 天津医科大学 ， 外科学， 2008， 硕士

【摘要】 目的肿瘤特异性细胞毒性T淋巴细胞(CTL)是体内抗肿瘤免疫最为有效的效应细胞,但其激活需要抗原呈递细胞(APC)处理并呈递相应的抗原。树突状细胞(DC)是目前已知功能最强的APC,可以激活初始T细胞和记忆性T细胞。DC可提供T细胞活化所需的共刺激信号,在体内外诱导CTL的生成。肿瘤组织匀浆、细胞裂解物包含全部抗原信息,用其致敏DC细胞,体外可以激发T细胞大量增殖,产生肿瘤特异性CTL。转化生长因子β(transforming growth factor-β,TGF-β)是肿瘤细胞分泌的功能最强的免疫抑制因子。TGF-β具有通过抑制肿瘤抗原呈递、淋巴细胞的杀伤作用等多种途径,负性调节机体对肿瘤的免疫应答和促进肿瘤生长的作用。我们通过用携带显性负相的TGF-βⅡ型受体基因的慢病毒载体感染人CTL,使其对TGF-β不敏感,为晚期前列腺癌的治疗探索一条新的治疗途径。方法我们用同一患者来源的DC、肿瘤组织匀浆、T淋巴细胞培养出肿瘤特异性CTL,然后以慢病毒为载体将TβRⅡ-DNglytk基因转染到肿瘤特异性CTL,从而去除了TGF-β对肿瘤特异性CTL的抑制作用。我们先用PC-3肿瘤细胞裂解物致敏健康人来源DC,体外刺激T淋巴细胞,获得对TGF-β不敏感肿瘤特异性CTL,并且通过流式细胞术,荧光抗体染色,Western—blot,MTT等方法对其进行了分析、鉴定。在此基础之上,我们将前列腺根治性切除术后标本移植到裸鼠身上,进行扩增,获得足量的肿瘤组织,用肿瘤组织匀浆作用于同一患者来源DC,然后刺激T淋巴细胞,成功获得了对TGF-β不敏感肿瘤特异性CTL,并经体内实验证实了对TGF-β不敏感肿瘤特异性CTL的有效性。结果本实验采用人重组IL-4、人重组GM-CSF、PC-3肿瘤细胞裂解物体外诱导PBMC成功培养出负载肿瘤抗原的DC细胞,以此致敏T淋巴细胞,获得肿瘤特异性细胞毒性T淋巴细胞;成功将TβRⅡDNglytk基因转入肿瘤特异性CTL细胞,并获得鉴定;转染对细胞状态、活性影响很小;体内、外实验验证转染后一定程度上去除了TGF-β对CTL杀伤肿瘤细胞的抑制。结论对TGF-β不敏感肿瘤特异性CTL培养成功及其体内、外实验研究表明:这一方法在肿瘤免疫治疗中具有潜在的应用前景,为肿瘤的过继免疫治疗提供了又一新的思路。更多还原

【Abstract】 ObjectivesCytotoxic T lymphocytes(CTL) are the most important effect cell in anti-tumor immune response in vivo. CTL must be activated through the presentation of appropriate antigen by antigen-present cells(APC). Dendritic cells(DC) are the potentest APC. As we known, they can activate not only naive T cells but memory T cell. Cytotoxic T lymphocytes(CTL) can be induced by DCs which can provide co-stimulating signal that is indispensable to activation of T cells. Cancer cells produce large amounts of TGF-β, which is a potent immunosuppressant . The high levels of TGF-βproduced by cancer cells have a negative effect on surrounding cells, including the host immune cells. As a result, cancer cells are able to escape the host’s immune surveillance program, leading to tumor progression and metastasis .Material and methodsThe present study involved isolation and generation of dendritic cells, CD4+ T cells, and CD8+ T cells from patients with prostate cancer. Next, We harvested tumor cells from patients with advanced prostate cancer undergoing radical prostatectomy to generate the primary cancer cell cultures but failed to subculture the cells. Because of the limited quantity of prostate tumor, we primed the CTL with PC3 cell lysates and dendritic cells. Tumor reactive CTL will be generated by isolate lymphocytes cocultured with dendritic cells in the presence of lysates of PC-3 cells and IL-2,IL-4,GM-CSF. Then, we will render these cells insensitive to TGF-βby transfection with the TβRⅡDN-tk construct. The control CTL will be transfected with the TRANSglytk vector. Then we performed adoptive transfer of autologous tumor-reactive TGF-βinsensitive CTL into these immuno-deficient mice bearing human prostate tumors. Finally, the effectiveness of tumor-reactive TGF-βinsensitive CTL in vivo were determined using FACS, immunofluorescence, western-blot, MTT and so on. ResultsThe mixed culture of lymphocytes and DCs made the autologous tumor-reacive CTL cells. Establish the efficacy of transfection using the lentivirus-based gene transfer method to generate TGF-βinsensitive CTL cells: Tumor reactive CTL were infected with the lentiviral particle containing TβRⅡDNglytk and TRANSglytk. Infection efficiency were assessed for V5-epitope using anti-V5-FITC. The results showed that adoptive transfer of tumor-reactive TGF-β-insensitive CTL in immunodeficient mice bearing xenograft tumor from human Prostate carcinoma can significantly suppress the growth of the xenograft tumor.ConclusionOur experiment made a foundation for further application of tumor-reactive TGF-βinsensitive CTL in adoptive immunotherapy of prostate tumors.更多还原