CD176 抗体介导的白血病免疫治疗的实验研究以及CD176 单链可变区的构建
文献类型：学位论文
| 作者 | 张哲 |
| 学位类别 | 硕士 |
| 答辩日期 | 2013-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 曹毅 |
| 关键词 | CD176抗原 白血病免疫治疗 噬菌体库 scFv 小分子抗体 |
| 其他题名 | Leukemia Immunotherapy Mediated by CD176 antibody & Construction of the CD176 scFv |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 白血病是一种严重危害人类健康的恶性肿瘤疾病，每年全世界有数十万人患此疾病。白血病的治疗方式主要以传统的放化疗，诱导治疗以及骨髓移植为主要方式，但是每种方式都有自己的局限性。目前，生物治疗成为一种新的，更有效的白血病治疗方式，其疗效已获得了肯定。 CD176（Thomsen-Friedenreich抗原，结构Gal1-3GalNAc1-R）是一种糖抗原。在正常细胞中，CD176抗原往往被唾液酸修饰而掩盖，并不暴露出来。但恶性肿瘤中，肿瘤细胞由于糖基化障碍，唾液酸修饰不足，造成CD176抗原暴露，暴露的CD176抗原因此可视为一种肿瘤抗原。CD176抗原表达于多种恶性肿瘤中，如乳腺癌、肠癌、胃癌、肺癌、肝癌、肾癌、淋巴瘤等，但不表达于成人的正常组织中。有研究显示，CD176抗原的表达与肿瘤的发展、侵润和转移有重要关系。我们之前的研究显示，CD176抗原在某些白血病细胞系中存在着高表达，并且通过CD176抗体的处理，能够诱导白血病细胞的凋亡。进一步研究，CD176抗体可能通过结合CD95等凋亡通路蛋白上的 CD176分子，诱导凋亡信号通路的激活，进而引起了白血病细胞的凋亡。 本课题中，我们对CD176抗体的体内治疗作用做了进一步研究，我们发现：(1)CD176抗血清治疗的CD176阳性白血病小鼠，其生存时间远优于对照组小鼠；（2）CD176抗血清治疗的小鼠，与对照组相比，其肝转移率显著降低（从对照组的90%下降到CD176抗体治疗组的30%），并且肝结节转移数目显著减少；（2）CD176抗血清治疗组小鼠的肝、肺、脾肿胀程度显著减轻，病理组织切片染色显示白血病细胞的数目也显著减少；骨髓涂片显示白血病的细胞数目也显著减少；（3）免疫组化显示CD176抗体能够诱导体内白血病细胞的凋亡。 在确定了CD176抗体对CD176+白血病治疗的疗效后，我们通过CD176抗原（aGP）免疫小鼠，提取小鼠脾中B细胞，构建小鼠脾B细胞cDNA文库，进而成功构建了抗CD176抗原的噬菌体文库。然后，我们通过ELISA筛选的方式，成功筛选到一株对CD176抗原有强结合能力的菌株 (命名为T48)。通过对T48中的scFv基因进行测序，我们成功克隆了T48中的scFv基因，并且构建了基于T48菌株中scFv基因的3个表达载体scFv-His tag、scFv-scFv和scFv-AP，用于以后该CD176抗体的进一步研究。 我们的研究表明，CD176抗体对CD176+白血病有着显著的治疗疗效，通过噬菌体库筛选的方式，构建高效的CD176小分子抗体的表达载体，为以后CD176治疗抗体的制备与新药研究，奠定基础。 |
| 英文摘要 | Leukemia is one of cancer that is severely harmful to human health, every year hundreds of thousands of people succumb to this disease worldwide. The therapy of leukemia mainly contains three ways: radiotherapy and chemotherapy, induction therapy and bone marrow transplantation, however, all of them have their restrictions and drawbacks. At present, the biotherapy is a novel and more effective way to cure the leukemia patients, and its treatment effect is increasingly admitted. CD176 （Thomsen-Friedenreich antigen，chemical structure is Gal1-3GalNAc1-R）is acarbohydrate antigens. In normal cells, CD176 is modified by sialic acid and masked. While in cancer cells, due to limited modification of sialic acid, it exposed and hence can be think as a specific antigen for cancer cells. CD176 exposes on many kinds of cancer cells, such as breast cancer cells, colon cancer cells, lung cancer cells and leukemia cells, but not on normal tissue cells. Prior research indicates that CD176 expression has a significant relationship with the infiltration, metastasis and development of cancer. Our prior research shows CD176 expression on many kinds of leukemia cells and CD176 antibody can induce the apoptosis of these kinds of leukemia cells. Additional research demonstrates that CD176 antibody might combine with the CD176 on the CD95 et al. proteinof apoptosis pathway, activating the apoptosis pathway and causing the apoptosis of leukemia cells. In this project, we have lucubrated the effect of immunotherapy of the CD176 antibody in vivo in experimental animal. we demonstrate that: (1) treatment with CD176 anti-serum, CD176+ leukemia mice’s survival time gets longer than other treatment control groups, (2) in compare to the other control groups, liver metastasis rate significantly decreasesin CD176 anti-serum treatment group ( from the 90% of control groups down to the 30% of CD176 anti-serum treatment group).CD176 anti-serum can effectively alleviate the dwelling degree of liver，lung, spleen and decrease the leukemia cells number in liver and spleen tissue. additionally, the leukemia cells number in bone marrow of mice in CD176 anti-serum treatment group also decrease, (3) CD176 antibody can induce the apoptosis of leukemia cells in vivo through the immunohistochemical analysis. In approval of the therapy effect of CD176 antibody to the CD176+ leukemia mice, we immunized mouse with CD176 antigen (aGP), constructing the cDNA library of B cells, and successfully constructing the phage library of anti-CD176 antigen. Then, we have successfully screened a clone (named T48) with a high affinity to the CD176 antigen through ELISA method. Afterwards, we clone T48 scFv gene and construct three expression vector, scFv-His tag, scFv-scFv, scFv-AP, in order to have a further understanding about the CD176 antibody to CD176+ leukemia. Our research data also demonstrate CD176 antibody has a significant therapeutic effect to the CD176+ leukemia. Therefore, It has the scientific and social meaning to construct an efficient-expressed CD176 expression vector. |
| 语种 | 中文 |
| 公开日期 | 2013-07-01 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7539]  |
| 专题 | 昆明动物研究所_分子病理学 |
| 推荐引用方式 GB/T 7714 | 张哲. CD176 抗体介导的白血病免疫治疗的实验研究以及CD176 单链可变区的构建[D]. 北京. 中国科学院研究生院. 2013. |
入库方式： OAI收割
来源：昆明动物研究所
浏览0
下载0
收藏0
其他版本
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。