| 题名 | Thomsen-FriedenreicThomsen-Friedenreich 抗原(TF,CD176)介导的白血病免疫生物治疗的实验研究 |
| 作者 | 易濒 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-05 |
| 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 | TF抗原 TF抗体 白血病免疫治疗 细胞凋亡 分子机制 |
| 其他题名 | A Study on Leukemia Immunotherapy Mediated by Thomsen-Friedenreich Antigen(TF,CD176) |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 白血病是危害人民健康、生命安全的主要恶性肿瘤之一，可发生于任何年龄，但是在儿童及35岁以下成人的恶性肿瘤中居第一位，死亡率较高。白血病是全身性的恶性肿瘤，传统化疗、诱导治疗和骨髓移植是主要治疗方法。但传统化疗副作用太大；诱导治疗对某些白血病不敏感；而骨髓移植受供体和费用的限制，应用不广。目前，研发新的白血病的生物治疗是重要方向，通过诱导癌细胞凋亡，从而用于白血病的生物治疗，有很好临床应用前景。 Thomsen-Friedenreich抗原（TF抗原，CD176, Galβ1-3GalNAc1-R）是MN血型决定簇的前体物质，为一隐性血型糖抗原。在正常细胞上，TF抗原被唾液酸及其它糖链掩盖，恶性肿瘤时，肿瘤细胞由于糖基化障碍，TF抗原暴露，暴露的TF抗原可视为肿瘤抗原。TF抗原在70-80%癌中都有表达，在白血病细胞上也有表达，但TF不表达于成人的正常组织。TF抗原的表达与肿瘤的发展、侵润和转移有关。以前的研究中我们发现TF抗体体外可以诱导TF抗原阳性的白血病细胞凋亡，本课题将研究TF抗体诱导TF阳性白血病细胞凋亡的机制及TF抗体对TF阳性白血病小鼠的治疗效果。 本研究发现：（1）流式细胞技术和免疫荧光表明大部分白血病细胞系都表达TF抗原，大部分细胞系表达CD95分子，部分白血病细胞系表达CD45、CD43、DR4和DR3分子；（2）共聚焦荧光显微镜观察发现白血病细胞表面CD95和DR4分子与TF抗原均有共表达，免疫共沉淀方法和酶联免疫吸附实验夹心法证实CD95, CD45, CD43和DR4分子是TF抗原的载体蛋白；（3）TF抗体诱导KG-1细胞凋亡后，应用基因芯片杂交实验发现，直接参与了细胞凋亡的诱导、执行和正向调控的20个基因表达量上调，q-RT-PCR验证得到同样的趋势；（4）富集度统计学分析发现CD95信号通路及DR3与DR4/5死亡受体通路与TF抗体诱导白血病细胞凋亡相关。我们推测，在白血病细胞上，TF抗体结合在细胞表面凋亡相关糖蛋（如：CD95、DR4等）的TF糖链上，激活凋亡通路，因此导致了TF阳性的白血病细胞凋亡。 我们亦通过体内实验研究是否TF抗体对TF阳性白血病小鼠有治疗效果。研究结果发现：（1）TF抗体能显著性降低白血病小鼠肝、脾肿大程度，经组织切片检查其治疗组白血病细胞数量明显降低；（2）统计检验结果表明TF抗体免疫治疗能显著性降低实验小鼠肝转移发生率（从对照组的90%到TF 抗体治疗组的30%），并且能显著性降低肝转移结节数量。卡方检验结果表明，与对照组相比，TF抗体尾静脉注射能显著降低实验小鼠肺转移的发生率；（3）骨髓涂片检测发现，对照组小鼠骨髓大概有50%白血病细胞，TF抗体处理能减少实验小鼠骨髓中白血病细胞（TF抗体处理组大概只有10%白血病细胞）；（4）TF抗体处理能在体内诱导白血病细胞凋亡；（5）Kaplan-Meier分析表明，TF抗体和TF抗原疫苗治疗均能显著性延长TF阳性的白血病小鼠的生存时间。 TF抗原表达于人类白血病细胞表面，但不表达于人类良性和正常组织，TF抗体处理对TF阳性白血病小鼠有明显的治疗效果，因此，TF抗原是一个很有希望的白血病免疫治疗的新靶点。 |
| 英文摘要 | Leukemia is one of the major cancers which harmful to people's health and safety, and it can occur at any age. However, among children and adults under the age of 35, the mortality of leukemia is in the first place. Leukemia is a systemic malignancy. Most types of leukemia are treated with conventional chemotherapy, induction therapy and bone marrow transplantation. However, the traditional chemotherapy has side effects; the induction treatment is insensitive to certain leukemia; the marrow transplant is limited due to the reason of donors and the expense. Thus, development of new biological treatments for leukemia is required. The biological treatments of leukemia by induction of apoptosis may have good prospects for clinical application. The Thomsen-Friedenreich antigen (TF antigen, CD176, Gal1-3GalNAc1-R) is the MN blood group determinants precursors, and is a recessive blood-group antigen. In normal cells, TF antigen is covered by sialinic acid and other sugar chain. In malignant tumors, the glycosylation barrier of the tumor cell result in the exposure of TF antigen, which may be regard as the tumor antigen. Approximately, 70-80% of carcinomas carry TF on their surface and leukemia cells also express TF. But TF is absent in normal and benign adult human tissues. The increased TF occurrence in cancer cells may be related to cancer progression, invasion, and metastasis.In a previous study, we observed that the TF antibody can induce the apoptosis of TF-positive leukemic cells in vitro. In this study, we investigated the mechanisms of apoptosis induced by TF antibody and whether TF antibody have a therapeutic effect on TF-positive leukemia mice in vivo. We found these results: (1) Flow cytometry analysis and immunofluorescence staining showed that almost all leukemia cell lines expressed TF antigen, most of the cell lines expressed CD95 molecule and some leukemia cell lines expressed CD45, CD43, DR4 and DR3. (2) We found that CD95 and DR4 are co-expressed with TF on the surface on defined leukemic cells observed by confocal microscopy and flow cytometry analyses. CD95, CD45, CD43 and DR4 are carrier proteins of TF in hematopoietic cells recognized by means of sandwich ELISA and co-immunoprecipitation. (3) In microarray analysis, 20 genes which are directly related to execution, induction or positive regμlation of apoptosis, were up-regμlated after the TF antibody treatment on KG-1 cell line. We use q-RT-PCR to validate and have the same trend. (4) The analysis of enrichment statistics demonstrated that CD95 signaling pathway and DR3 and DR4/5 death receptors pathway were mostly relevant to the apoptosis induced by TF antibody. We hypothesized that TF antibody binds the TF carbohydrate structure present on apoptosis-associated glycoproteins at the cellular surface such as CD95 and DR4, activate apoptotic pathways, and consequently result in the apoptosis of TF-positive cells. We also investigated whether TF antibody have a therapeutic effect on TF-positive leukemia mice in vivo. Our results showed (1) TF antibody treatment could significantly reduce the swelling degree of liver and spleen of leukemia BALB/c mice compared to control leukemic BALB/c mice. Histopathology analysis showed the number of metastatic leukemic cells in TF antibody-treated leukemia mice were evidently decreased. (2) Statistics analysis showed TF antibody immunotherapy resulted in a significant decline in the overall incidence of liver metastasis (from 90% in the control group to 30% in the TF antibody-treated group) and significantly decreased the number of liver metastasis nodules. Chi-square test demonstrated that TF antibody i.v. treatment could significantly decrease the incidence of lung metastasis compared to control leukemia BALB/c mice group. (3) The bone marrow smear of four groups mice showed that anti-CD176 antibody treatment could reduce the amount of metastatic cell of bone marrow. Anti-CD176 antibody treated mice had about 50% metastatic leukemia cells compared to the 10% of control leukemic BALB/c mice. (4) TF antibody treatment could induce the apoptosis of leukemic cell in vivo. (5) Kaplan-Meier analysis of in vivo TF antibody and TF vaccine (aGP) immunotherapy experiments showed that immunotherapy with TF antibody and aGP provided leukemia mice bearing TF-positive leukemic cells with a significant survival advantage in comparison to control leukemia BALB/c mice group. TF is expressed at the surface of human leukemic cells, but is almost absent in normal adult human tissues. TF antibody have a significantly therapeutic effect on TF positive-leukemia mice. Thus, TF may be a promising target for anti-leukemia immunotherapy. |
| 语种 | 中文 |
| 公开日期 | 2012-09-25 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7125]  |
| 专题 | 昆明动物研究所_分子病理学 |
| 推荐引用方式 GB/T 7714 | 易濒. Thomsen-FriedenreicThomsen-Friedenreich 抗原(TF,CD176)介导的白血病免疫生物治疗的实验研究[D]. 北京. 中国科学院研究生院. 2012. |
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