【作者】 徐彤；

【导师】 郝希山；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2003， 硕士

【摘要】 对于外周血干细胞移植支持下大剂量化疗治疗的实体瘤患者，治疗后免疫功能重建并清除残留病灶是关键问题。利用基因治疗和免疫治疗手段作为防止肿瘤复发的有效武器有着巨大希望。Fas抗原(APO-1／CD95)与Fas配体(FasL)组成的Fas系统，在将凋亡信号转导入细胞内发挥重要作用。效应细胞FasL结合靶细胞Fas抗原后，在数小时内通过细胞凋亡途径使靶细胞死亡。近年来很多研究发现Fas在多种细胞表面均有表达，而FasL表达只限于少量细胞类型，诸如淋巴细胞、免疫特权器官的细胞和多种恶性肿瘤细胞，同时已证实表达FasL的肿瘤周围激活的Fas阳性淋巴细胞凋亡异常增加。另一方面，肿瘤的发生在很大程度上是因为转化的细胞不能正常凋亡所致。因此，研究如何抑制肿瘤的Fas反击(counterattack)和如何促进肿瘤细胞的凋亡将为肿瘤治疗提供新的途径。通过对FasL功能的深入研究将有助于充分认识FasL基因治疗的潜力，甚至提供一种有效的治疗措施。 根据早期的文献，通过表达Fas配体对Fas介导凋亡途径的抵抗可能会使多种肿瘤对机体免疫系统进行优先攻击或反击。从概念上讲，消除肿瘤的Fas反击(counterattack)是肿瘤免疫治疗的一个令人兴奋的有效目标。我们将把表达Fas的T淋巴细胞和表达FasL的肿瘤细胞的体外相互作用作为本项目的研究对象，利用反义FasL核酸技术拮抗肿瘤细胞的FasL表达或功能，以达到消除肿瘤的Fas反击并延长TIL寿命的目的，这将最终促进免疫功能重建，从而提高抗肿瘤的效果。天津医科大学硕士研究生学位论文 第一部分肿瘤细胞逃逸Fas系统介导的免疫杀伤的研究 为研究结肠肿瘤细胞系表达FasL逃逸免疫监视的可能，研究中采用了敏感的免疫组织化学SABC法观察结肠癌细胞系和JurkatT淋巴细胞系Fas与FasL的表达，为癌细胞的Fas和FasL的功能提供形态学依据。结果提示SW620结肠癌细胞FasL呈阳性，阳性物质定位于细胞膜及胞质，胞核为阴性反应;JurkatT淋巴细胞FasL呈阳性。JurkatT淋巴细胞呈Fas呈阳性，阳性物质定位于细胞膜及胞质，胞核为阴性反应;而结肠癌Sw620细胞Fas呈阴性。我们采用非放射性细胞毒分析，测定sw620结肠癌细胞与Jurkat靶细胞共培养后LDH释放值检测效应细胞的杀伤效应。同时测定靶细胞自发释放值、靶细胞最大释放值、效应细胞自发释放值、体积校正值和培养基背景值。应用如下公式计算杀伤效应:杀伤效应(%)=(实验值一效应细胞自发释放值一靶细胞自发释放值)/(靶细胞最大释放值一靶细胞自发释放值)x 100。结果提示在结肠癌细胞系表达功能性的FasL，能杀伤Fas阳性的JurkatT淋巴细胞。 第二部分人FasL反义真核表达载体的构建和鉴定 为了研究抑制肿瘤的Fas反击(contera沈昵k)的可行性，一种新的重组真核表达载体peDNA3 .1~hFL一s被构建。将人FasL cDNA亚克隆入人巨细胞病毒(cMv)启动子控制基因表达质粒peDNA3 .1乃al位点构建真核表达载体PcDNA3.1-hFL一ASo PcDNA3.1-hFL一S酶切鉴定结果显示分别为1.85kb和4.55kb片段。基因序列分析确定人FasL cDNA被正确插人载体，证实核昔酸 2 天津医科大学硕士研究生学位论文序列正确。重组真核表达载体 pCDNA3．lhFL ots的成功构建使进一步研究FasL的功能成为可能，甚至提供一种有效的治疗措施。更多还原

【Abstract】 For solid tumor patients undergoing high-dose chemotherapy sustained by peripheral blood stem cell transplantation (PSCH), it is crucial to improve immune reconstitution and get rid of remaining cancer. Gene therapy and irnmuotherapeutic approaches hold great promise as effective weapons against the recurrence of tumor. The Fas/Fas ligand (FasL) system plays an important role in the transduction of apoptotic signal into the cells. Within several hours after the engagement of target cells Fas by effector cells FasL, target cells undergo cell death by way of the Fas-mediated apoptosis. In recent years, numerous studies have demonstrated that Fas is expressed on the surface of cells of various types, whereas FasL expression is restricted to a small number of cell types, such as lymphocytes, cells of the immune-privileged organs and many types of malignant tumor cells. Meanwhile, evidence has pointed to an abnormal increase in apoptosis among activated Fas-positive lymphocytes, mainly in the periphery of the FasL-expressing tumors. On the other hand, to a great extent, the occurrence of tumor is due to the fact that the converted cells can not undergo a normal process of apoptosis. Therefore, a research about how to disarm the tumor’s Fas counterattack and how to promote tumor cell apoptosis will provide new approaches to the therapy of tumors. A better understanding of FasL function will help to fully realize the potential of the FasL gene therapy and even offer a potential therapeutic intervention.According to earlier literature, resistance to apoptosis through the Fas receptor pathway coupled with expression of the Fas ligand might enable many cancers to deliver a pre-emptive strike or counterattack against the immune system. Disarmingthe Fas counterattack is a conceptually appealing and exciting potential goal for tumor immunotherapy. Our research is about the interaction in vitro between T cells that express Fas and tumor cells that express FasL. The approach of this project is to block the expression or function of FasL on the tumor cell by antisensing FasL, with a view to abrogate the Fas counterattack and prolong TIL’s life span, which will ultimately regulate immune recovery and enhance antitumoral efficacy.Part I The study on tumor cells escaping from Fas-mediated apoptosisTo investigate the possibility of colon cancer cells escaping from immune surveillance through the expression of FasL on tumor cells, a study was undertaken by using immunohistochemical SABC method. The expression of Fas receptor and Fas ligand in SW620 colon carcinoma cell line and Jurkat T cells was observed so as to supply morphological evidence for the functions of Fas receptor and Fas ligand. It was shown that the Fas ligand of colon carcinoma SW620 cells was positive. The positive substances were distributed in the cell membrane and cytoplasm while the nuclei of the cells were negative, and the Fas ligand of Jurkat T lymphocytes turned out to be positive. Meanwhile, it was also shown that the Fas receptor of Jurkat T cells was positive. The positive substances were distributed in the cell membrane and cytoplasm while the nuclei of the cells were negative, and the Fas receptor ofcolon carcinoma SW620 cells was negative. In an effort to examine the cytotoxicity of effective cells, we adopted CytoTox 96 Non-Radioactive Cytotoxicity Assay to measure LDH releasing value after the SW620 cells were co-cultured with the Jurkat T lymphocytes. At the same time, we also measured the values of Effector Cell Spontaneous LDH Release, Target Cell Spontaneous LDH Release, Target Cell Maximum LDH Release, Volume Correction Control and Culture Medium Background. Then the following formula was applied in the calculation of percentcytotoxicity: Cytotoxicity (%) = (Experimental- Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous) X 100. The result indicates that FasL expression in the colon carcinoma SW620 cells could inversely induce apoptosis of Fas-expressing Jurkat T lymphocytes.Part更多还原