【作者】 杜耀武；

【导师】 马远方；

【作者基本信息】 河南大学 ， 免疫学， 2007， 硕士

【摘要】 食管癌(Esophageal carcinoma, EC)是世界六大恶性肿瘤之一,其主要流行病学特征是显著的地域性分布差异,高低发区发病率相差高达500倍,并且预后极差,中晚期患者5年生存率仅为10%左右。在中国食管癌患者以食管鳞状上皮细胞癌(Esophageal squamous cell carcinoma, ESCC)为最常见。食管癌的治疗方法及效果对食管癌防治致关重要,近年来,随着食管癌的发病机理和治疗研究的进展,人们认识到细胞凋亡机制的异常在食管癌发生和抗肿瘤治疗中起重要作用,研究发现很多抗肿瘤治疗方法是通过诱导肿瘤细胞凋亡发挥作用。因此,寻找能够诱导食管癌细胞凋亡的活性物质,通过激发食管癌细胞的凋亡途径,达到治疗食管癌的目的是食管癌防治的有效方法之一。肿瘤坏死因子相关的凋亡诱导配体(TNF-related apoptosis inducing ligand, TRAIL)是1995年Wiley等发现的TNF超家族成员,能够诱导多种肿瘤细胞凋亡而对大多数正常细胞无影响。TRAIL通过与肿瘤细胞膜上的相应受体结合诱导细胞凋亡,目前发现TRAIL有5种类型的受体,其中TRAIL受体1(死亡受体4 death receptor 4, DR4)和TRAIL受体2(死亡受体5 death receptor 5, DR5)胞内段含有死亡结构域,能够传递凋亡信号,其他三种受体:TRAIL受体3(DcR1)、TRAIL受体4(DcR2)和OPG(Osteoprotegerin)可阻断TRAIL诱导的凋亡,称为“诱骗”受体。与TNF和FasL不同,TRAIL能够诱导多种肿瘤细胞和病毒感染细胞凋亡,但对正常细胞无影响,TRAIL这一特性使TRAIL有望成为特异的抗肿瘤新药。然而随着研究的深入,发现有些重组TRAIL(rhTRAIL)对正常肝细胞等也有一定的凋亡作用,这使人们对TRAIL临床应用的安全性产生了疑虑,加之TRAIL受体的多样性影响了TRAIL诱导细胞凋亡作用的发挥,也一定程度上限制了TRAIL的临床应用。实验发现一些抗TRAIL受体1和受体2(DR4、DR5)的抗体,也有诱导细胞凋亡的作用,由于在生理条件下TRAIL与DR5的亲和力明显大于DR4,因此,在TRAIL诱导细胞凋亡过程中DR5是起主要作用的受体。以TRAIL诱导凋亡的主要受体DR5为靶点,研制能激发肿瘤凋亡的抗DR5的单克隆抗体成为TRAIL临床研究的新方向,能诱导肿瘤细胞凋亡的抗DR5单抗有可能替代TRAIL有效地用于肿瘤临床治疗。目的:制备出能够诱导肿瘤细胞凋亡抗人DR5的单克隆抗体,探讨该抗体对食管癌细胞系及食管上皮细胞系的诱导凋亡的分子机制,并就此抗体进行了初步的抗肿瘤作用的动物实验,为下一步的功能抗体的人源化改造及临床实验研究奠定基础,以早日实现此功能抗体的临床应用。方法:提取纯化可溶性人DR5(soluble DR5,sDR5)胞外段,用sDR5免疫BALB/c小鼠,将小鼠脾细胞与NS-1细胞在50%PEG条件下,通过细胞融合技术,获得了诱导肿瘤细胞凋亡的抗人DR5的单克隆抗体-命名为mDRA-6,mAb采用Protein G亲和层析柱纯化,免疫印迹检测其特异性。食管鳞癌细胞表面DR5表达水平采用流式细胞仪检测;单克隆抗体对食管癌细胞的杀伤活性用MTT法检测、对食管鳞癌细胞系的凋亡作用采用Hoechst 33258染色荧光显微镜下观察细胞形态变化,采用JC-1单染及Annexin V-FITC/PI双染流式细胞术定量分析技术对食管鳞癌细胞系的凋亡进行定量分析。利用免疫印迹技术(Western blotting)检测凋亡细胞的Caspase8、Caspase3、Caspase9及Bid、bax、bcl-2、cytochrome c表达情况。建立食管癌磷癌EC109荷瘤Scid小鼠模型,腹腔注射mDRA-6观察肿瘤的生长及消退情况。结果:1.用不表达DR5的NS-1骨髓瘤细胞融合的方法,制备诱导肿瘤细胞凋亡的抗人DR5的单克隆抗体(命名为mDRA-6),该抗体能与DR5特异性结合。2. DR5在食管鳞癌及转化食管细胞系表面的表达:在所检测的4种食管鳞癌及转化食管细胞系表面均有DR5的表达,人食管鳞癌低分化细胞系EC9706、高分化细胞系EC109细胞系DR5的表达为91.31%、96.56%,食管永生化细胞NE6的DR5的表达表达45.81%,食管上皮细胞系NEC的DR5的表达5.64%。食管鳞癌细胞系DR5表达水平最高,食管永生化细胞次之,食管上皮细胞最低。3. mDRA-6单抗对食管鳞癌细胞的细胞毒作用:MTT法检测显示,mDRA-6在20μg/ ml浓度作用24小时,对食管鳞癌EC9706、EC109细胞系的抑制率分别为56.53%、68.82%,对食管永生化细胞NE6的抑制率为26.82%,而对食管上皮细胞系NEC细胞几乎无有作用,仅为4.56%。有此可知mDRA-6对细胞的生长抑制与细胞表面的DR5表达呈正相关性。4. mDRA-6单抗对食管鳞癌细胞的凋亡作用:在荧光显微镜下mDRA-6导致细胞呈现典型细胞凋亡的形态特征; Annexin V-FITC/PI双染流式细胞术检测显示, mDRA-6在20μg/ ml浓度作用EC9706细胞6、12、24小时,凋亡率分别为20.58%、39.90%、50.09%,作用于EC109细胞6、12、24小时凋亡率分别为27.47%、33.28%、57.46%。线粒体膜电位试剂盒(JC-1)检测显示EC9706细胞6、12、24小时,凋亡率分别为15.71%、24.40%、44.93%,EC109细胞凋亡率分别为36.60%、40.48%、52.31%。5.免疫印迹技术(Western blotting)检测凋亡细胞的Caspase8、Caspase3、Caspase9及Bid、bax、bcl-2、cytochrome c表达情况:显示mDRA-6诱导人食管鳞癌细胞凋亡的分子机制是通过死亡受体DR5,激活和启动外源性细胞凋亡途径,进而又激活内源性细胞凋亡途径,最终引起肿瘤细胞的凋亡。6.注射mDRA-6的荷瘤Scid小鼠的肿块与对照组相比较小,证明mDRA-6具有明显的抗肿瘤作用。结论:1.获得了诱导肿瘤细胞凋亡的抗人DR5的功能性单克隆抗体mDRA-6。2.在所检测的4种食管鳞癌细胞及食管上皮细胞系、食管永生化细胞表面均有DR5的表达,但不同细胞表面DR5的表达水平存在着很大的差异。3.mDRA-6能够诱导人食管鳞癌细胞系EC9706、EC109的凋亡,主要通过激活死亡受体细胞凋亡信号传导途径发挥作用。4.mDRA-6对荷瘤Scid小鼠具有很好的抗肿瘤作用,为今后此功能抗体的人源化改造及临床实验研究奠定了基础。更多还原

【Abstract】 Esophageal carcinoma (EC) is one of the sixth malignant tumors worldwide. EC is featured by geographic variation in incidence and poorest prognosis. Epidemilogical studies have indicated that incidence in highest region of EC could be 500-fold higher than lowest area of EC. The five-year survival rate for EC is only 10% for advanced EC patients. Patients with Esophageal squamous cell carcinoma(ESCC) are common in China.The present treatment modalities of EC comprise combination therapy, e.g. surgery, radiotherapy, chemotherapy and biological therapy. However, the efficiency of modalities above is still ominous and we need to explore effective ways to treat EC. Neoplastic growth results from loss of balance of cell growth and apoptosis. A large body of evidence has shown that aberrant mechanisms of apoptosis contribute to pathogenesis of EC and thus many anti-malignant treatments implicate apoptotic pathway. Accordingly, the identification of agents which can trigger apoptosis of EC is necessary, and this will promise to be one of effective treatments for EC.TRAIL (TNF-related apoptosis inducing ligand) was identified by Wiley in 1995 ,which is one of members of TNF super-family and to be able to induce apoptosis in tumor cells with no harmful effects on normal cells. TRAIL can bind to death receptors on cell membrane following by apoptotic pathway cascades activation. The receptors binding to TRAIL fall into five types, in which TRAIL receptor 1/DR4 (death receptor 4) and TRAIL receptor 2/DR5 (death receptor 5) posses the intracellular death domain contributing to apoptotic activation. However, other three types of TRAIL receptors, i.e. TRAIL receptor 3 (DcR1), TRAIL receptor 4 (DcR2) and OPG (Osteoprotegerin) were revealed to be decoy receptors due to lacking intracellular death domain. Where TRAIL is different from TNF and FasL in TRAIL inducing apoptosis in tumor cells and cells infected by virus ,as well as without harm to normal cells. In this regard, TRAIL can be developed as an ideal anti-tumor substance. Conversely, other investigations revealed that some recombinant human TRAIL (rhTRAIL) can induce apoptosis in normal liver cells, which gives rise to its safety in clinical application. In addition, multi-types of TRAIL receptor could reduce activity of TRAIL and hinder its clinical application. Further studies have found that monoclonal antibody against TRAIL receptor 1 and 2 (DR4 and DR5) has the abilities of TRAIL and can induce apoptosis in tumor cells. In normal physiological conditions, DR5 is the main functional receptor due to its binding affinity of TRAIL stronger than DR4. Currently, monoclonal antibody targeting DR5 receptor with apoptotic activity has great promise to become choice of effective anti-tumor treatment and has drawn much attention in this field.Objectives: To procure DR5 monoclonal antibody with apoptotic activity in tumor cells as well as to determine its effects and mechanisms on esophageal tumor cells, immortal esophageal epithelial cells and Scid tumor mouse model inoculated with esophageal tumor cells.Methods: Mouse monoclonal anti-DR5 antibody, designated as mDRA-6, was prepared by BALB/c mouse immunized by sDR5,when mouse spleen cell was fused with myeloma NS-1 cells at presence of 50% PEG (polyethylene glycol). specific binding of mDRA-6 by Western blot analysis, and mDRA-6 binding cell surface DR5 by flow cytometry. mDRA-6 was purified by protein G affinity chromatography. Flow cytometry was used to describe the DR5 distribution on esophageal tumor cells and immortal cells. Suppression of cell growth was determined by MTT assay, Hoechst 33258 staining was used to determine changes of cell morphology under fluorescence microscope, and JC-1 single staining and Annexin V-FITC/PI double staining were used to quantify apoptotic outcome after mDRA-6 exposure. Caspase 8, caspase 3, caspase 9 as well as Bid, bax, bcl-2 and cytochrome c were analyzed by Western blotting analysis after mDRA-6 treatment. Scid tumor model inoculated with esophageal cancer cells EC109 was injected with mDRA-6 in abdominal cavity to value mDRA-6’s effects on tumor growth.Results:1. Mouse monoclonal DR5 antibody (mDRA-6) with specific ability binding to DR5 is generated by cell fusion of myeloma NS-1 cells with BALB/c mouse spleen cells follwing sDR5 immunization.2. DR5 cell membrane expression is distinct on cells with different biological behavior, which are 91.31%, 96.56%, 45.81%, 5.64% on a poorly differentiated ESCC cell line EC9706,a well differentiated ESCC cell line EC9706, esophageal immortal cells NE6 and NEC, respectively. 3. MTT analysis showed that esophageal cancer cell growth is suppressed after mDRA-6 treating at 20μg/mL for 24 hrs, i.e. EC9706 (56.53%), EC109 (68.82%), while immortal esophageal epithelial cell after mDRA-6 treating showed little growth inhibition, i.e. NE6 (26.82%) and NEC (4.56%).4. Esophageal cancer cells EC9706 and EC109 displayed typical apoptotic morphology after mDRA-6 treatment at 20 ug/ml for 6 hrs, 12 hrs and 24 hrs when observed under fluorescence microscope. The apoptotic frequency was 20.58%, 39.90%, 50.09% for EC9706 and 27.47%, 33.28%, 57.46% for EC109 at 6 hrs, 12 hrs and 24 hrs, respectively. Mitochondria membrane potential was 15.71%, 24.40%, 44.93%for EC9706 and 36.60%, 40.48%, 52.31% for EC109 at the above time points, respectively.5. Apoptotic pathway induced by mDRA-6 in esophageal cancer cells is initiated by DR5 and mDRA-6 binding followed by exogenic and later endogenic cell apoptotic cascades activation as revealed by caspase 8, caspase 3, caspase 9 and Bid, bax, bcl-2, cytochrom c by western blotting, respectively.6. Tumor growth in Scid tumor model is suppressed markedly by mDRA-6 injection in abdominal cavity compared with control group.Conclusions:1. We procured a anti-DR5 monoclonal antibody with apoptotic activity derived from BALB/c mouse which is designated as mDRA-6.2. DR5 expression on esophageal cancer cells (EC9706 and EC109) is strikingly higher than immortal esophageal epithelial cells (NE6 and NEC).3. mDRA-6 induced apoptosis in esophageal cancer cells EC9706 and EC109 through binding to DR5 followed by apoptotic cascades activation, i.e. caspase 8, caspase 3, caspase 9 and Bid, bax, bcl-2, cytochrom c.4. In vivo Powerful anti-tumor effects of mDRA-6 on Scid tumor model paved grounds for mDRA-6 humanization and its clinical application.更多还原