【作者】 袁时芳；

【导师】 李开宗； 张英起；

【作者基本信息】 第四军医大学 ， 外科学， 2002， 博士

【摘要】 尽管化疗、放疗等有效的抗肿瘤治疗使癌症患者生存率不断提高，然而，大多数癌症转移病人因不能被治愈而死亡。因此，探索有效的治疗肿瘤的新途径十分必要。与其它治疗肿瘤的方法不同，肿瘤疫苗它通过激活机体自身的免疫系统来达到治疗的目的，从而具有高度特异性，其中DNA疫苗以其简单而独特的免疫方式和良好的动物免疫效果而倍受关注。与传统疫苗不同，DNA疫苗它能诱导出其表达的外源蛋白的特异性CTL，这正是预防和治疗肿瘤的关键。 人MUC1基因的编码产物Mucin是一种Ⅰ型跨膜蛋白，它通常位于腺上皮细胞的顶表面。研究发现，MUC1在乳腺癌等多种肿瘤中异常表达且由于糖基化不全，导致新的抗原肽表位暴露。因此，MUC1是肿瘤主动特异性免疫治疗理想的靶分子。pcDNA3．1(+)真核载体带有CMV启动子和SV40多聚腺苷信号序列，能促进外源基因高效转染和表达。为研究MUC1核酸疫苗的特异性抗肿瘤作用，我们构建了含人MUC1全长cDNA序列的真核表达载体pcDNA3．1(+)-MUC1，并全面评价了该基因疫苗的免疫学特性及GM-CSF免疫佐剂作用。另外，为探讨MUC1在肿瘤诊断和免疫治疗中的意义，我们采用 第四军医大学博卜学位论文 免疫组化方法检测了 MUC在不同肿瘤组织中的表达。 为此，首先提取质粒酶切回收MUC目的基因，克隆入pGEM习 刁后双 酶切鉴定质粒。DNA测序分析与己知的人 MUC。DNA序列相符。序列分析 鉴定后，将 MUC目的基因再亚克隆入 pCDNA3．l（＋卜 构建含人 MUC全长 CDNA序列的真核表达载体 pCDNA3．l（＋）MUC，并进行双酶切鉴定。 接着，将 pcDNA3．l什）－MU山电穿孔法转染 COS刁细胞，瞬时转染后 56h， 活细胞兔疫荧光染色，荧光显微镜观察发现，经重组质粒转染的COS刁细胞 膜表面可见明亮荧光，而 pCDNA3二什）空载体转染的细胞内未见荧光，表明 pcDNA3．l什）－MUC在 COSj细胞中以细胞膜分子形式表达。流式细胞仪检 测进一步证实了表达结果。 随后采用股四头肌注射法将质粒 pCDNA3．IMUCI肌注免疫雌性 BALB儿 小鼠，3周 1次，共 3次，每次基因免疫后 1，3，sd，皮下注射 GM．CSF 00ul （lug／100u人最后一次基因免疫后第3周，接种表达MUCI的EMT6小鼠 乳腺癌细胞及H22肝癌细胞，进行保护实验。两周后观察、记录肿瘤的生长 情况及荷瘤小鼠的存活期。采用标准 4h’七r释放法检测 CTLs杀伤功能；免 疫组织化学法检测 MUC特异性的体液免疫应答；采用直接免疫荧光法，流 式细胞仪检测T淋巴细胞表型。取小鼠瘤体组织，制备组织切片，HE染色镜 检，观察肿瘤的组织细胞结构。 肿瘤细胞接种后 45天，加 GM－CSF佐剂预防组、MUO预防组、质粒 pcDNA3．l＋GM－CSF对照组、pcDNA3＊对照组，EMT6月瘤大小分别为 （145土门．8）mm3、（250t24．3）mm3、（538t43＊）mm3、（596t4．2）mm3： H22月瘤大小分别为（293t26．3）mm’、（547t58＊）＊＊3、（1207ti48） mm‘、门 59）mm3。肿瘤1长曲线结果表明，与pcDNA3．l对i组相 比，MUC预防组 EMT6、H22肿瘤生长受到明显抑制（p＜0．05人 GM－CSF 佐剂有增强＊＊CI抗肿瘤生长作用…叼刀5人瘤重和病理学检查两个指标， 结果同样显示，MUC DNA疫苗及加 GM－CSF佐剂组与对照组的比较，抗 ＊MT6、H22月瘤组织生长均有显著性差异（p＜0＊5）。 杀伤实验结果表明，在不同的效靶比下，MUC ICTL对 EMT6靶细胞杀 伤率不同，100：l时最高，达 68．5％。预防组、加佐剂组与对照组对 EMT6、 4 第凹车医大学博1：学位论义 ＊22靶细胞的杀伤率相比均具有显著性差异（P＜0刀5）。 将 MUC基因疫苗免疫后小鼠血清作一抗，免疫组织化学法检测结果显 示，人乳腺癌组织 MUC均呈阳性染色，证实其可在小鼠体内产生特异性的 体液兔疫应答。结果表明外源目的基因在体内有效表达。 淋巴细胞表型检测结果显示，与 pCDNA3入对照组相比，MUC基因疫苗 预防组＊m升高具有显著性差异（k0刀5），＊04差别不显著：加＊M．CSF 佐剂预防组与单独 MUC 疫苗预防组相比，CD4 升高具有显著性差异 （P叱刀5），＊＊8差别不显著。 不同肿瘤组织及其癌前病变组织中的 MUC的表达结果表明，乳腺癌、 甲状腺癌、胃癌、肝癌组织与其良性病变组织及正常组织之间存在显著性差异 o＜0刀1X癌组织中＊＊CI阳性表达者为深棕色及棕黄色颗粒。不同肿瘤组织 分布定位不问。可位于细胞膜上、胞浆内及胞核。 综卜更多还原

【Abstract】 The survival rate of patients with cancer has improved, with the advent of effective anticancer treatments such as chemotherapy and radiotherapy. However, the majority of patients with metastatic disease will not be cured by these measures and will eventually die of their disease. New and more effective treating methods are required urgently. Differing from other measures, theraputic tumor vaccine could induce specific antitumour response by stimulating immune system. DNA vaccine is an attractive tool for vaccination studies because of its simple and unique given methods and substantial immune efficacy. It can induce not only specificantibodies production, but also specific cytotoxic T lymphocytes in animal experiments. This is the key point for prevention and treatment of cancer.Polymorphic epithelial mucin, encoded by the MUC1 gene, is a type I transmembrane protein, which presents at the apical surface of glandular epithelial cells. It is over-expressed and aberrantly glycosylated in many carcinomas resulting in an antigenically distinct molecule and a potential target for active specific immunotherapy. pcDNA3.1(+)vector, which has both CMV promoter and SV40 polyadenylation signal, can efficiently enhance transfection of foreign gene and then expression of recombinant protein. To study the specific anticancer role of MUC1 DNA as a vaccine, we constructed a eukaryotic expression vector pcDNA3.1(+)-MUCl containing the coding region of human full length MUC1 gene , and discussed immunological properties of MUC1 mucin and the efficacy tumour rejection induced by MUC1 DNA vaccine with and without GM-CSF adjuvant. Another, to study the changes expressions of MUC 1 in different tumor tissues and its clinical implications, a detailed immunohistochemical analysis of MUC1 protein expression was performed with MUC1 monoclonal antibody.First, The MUC 1 gene was obtained by cutting out the MUC 1 cDNA using Hindlll and Xhol and it was ligated into a clone vector pGEM-3zf. After identification and sequenthing , the gene was subcloned into eukaryotic expression vector pcDNA3.1(+). Restriction analysis and DNA sequencing showed that the recombinant plasmid contained the coding region of human full length MUC1 gene.The eukaryotic expression vector containing the MUC1 gene was succesfully constructed.Then, the recombinant pcDNA3.1(+)-MUCl was transfected into COS-7 cell by electroporation. After 56h transfection, Immunoflourescence and FCM were used to detect MUC1 gene expression in COS-7 cells. Fluorescence could been seen obviously in the transfected COS-7 cells membrane, whereas not in untransfected cells . Transfection experiment verified that MUC1 gene could be expressed in COS-7 cells and MUC 1 is a transmembrane protein molecule.Female BALB/c mice were immunized intramuscularly with 100 microgram MUC1 cDNA 3 times at 3-weekly intervals,. On 1, 3 and 5 days after Intra-muscular immunization, GM-CSF lOOul (lug/100ul) was given s.c. 3 times. Three weeks after the last immunization tumor challenge experiments were performed by using MUC1 expressing tumor cell line EMT6 ,H22. Tumor growth inhibition and body protection was observed two weeks later. Both humoral and cell-mediated MUC1-specific immune responses were detected with Immunohistochemical staining and 51Cr assays of CTLs, respectively. T-lymphocyte phenotype was detected by FCM. Histological analysis of tumor tissue was carried out with HE staining.After 45d of challenge experiments, the volume of EMT6 tumor in MUClcDNA, MUClcDNA+GM-CSF, pcDNA3.1(+), and PcDNA3.1(+)+GM-CSF group was (145?3.8)mm3, (250?4.3)mm\ (538?3.6)mm\ (596?8.2)mm3, respectively; the volume of H22 tumor in MUClcDNA, MUClcDNA +GM-CSF, pcDNA3.1 (+), pcDNA3.1 (+)+GM-CSF group was(293?6.3)mm3^ (547?8.6)mm3> (1207?48)mm\ (1220?59)mm3 , respectively. Compared with pcDNA3.1(+) control group, EMT6 tumor as well as H22 tumor growth in mice of MUClcDNA group were suppressed obviously(p<0.01). Co-delivery of GM-CSF adjuvant enhanced the antitumour immunotherapeutic effects.更多还原