【作者】 杨娟；

【导师】 沈关心；

【作者基本信息】 华中科技大学 ， 免疫学， 2011， 博士

【摘要】 [目的]本课题设计将姜黄素作为抗肿瘤药物在光照条件下激发成为光敏剂,通过修饰后偶联特异性的抗TfR抗体协同发挥双向抗癌效应。为了延长药物的半衰期,进一步探索姜黄素的光学特性,比较了TfR抗体与光敏剂联合应用的不同方法,检测其偶联效率及体外功能,探讨作用机制,并据此设计制备纳米化中药光敏剂-抗TfR单抗偶联物,为肿瘤的多重定位显像与靶向治疗提供实验依据。在此基础上还分析了从同一杂交瘤细胞株中扩增出的三条轻链基因,为实现抗TfR基因工程改造与基因工程抗体靶向偶联物抗瘤效应研究奠定基础。[方法]1.抗TfR单抗-偶联物的制备及其抗瘤效应测定(1)研究姜黄素光敏特性及荧光显微镜观察肝癌细胞内吞Cur；(2)以碳二亚胺为缩合剂制备Cur-Ab免疫偶联物,间接ELISA测定偶联物抗体效价,MTT法测定其IC50；(3)光敏化姜黄素-抗TfR单抗靶向性纳米粒的制备及效应测定：1)薄膜法和注入法制备脂质体,色谱法测定其包峰率；2) TfR mAb修饰的姜黄素脂质体的制备；3)扫描电镜观测脂质体姜黄素外形及粒径大小；4)FCM检测Cur-NP-PEG-Ab与肝癌细胞的结合率；5)FCM检测Cur-NP-PEG-Ab在光照和非光照情况下对肝癌细胞增殖/凋亡,周期等的影响；6)分别由DCFH-DA荧光探针和罗丹明染色试剂盒检测线粒体电位和细胞活性氧的变化。2.抗TfR单抗轻链基因分析(1)采用PCR法从分泌抗TfR单抗杂交瘤细胞中扩增抗体可变区序列；(2)通过IMGT和NCBI数据库在线分析VH和VL基因序列；(3)经由Insight II软件模拟抗TfR单抗Fabs段分子结构。[结果]1.抗TfR单抗-偶联物的制备及其抗瘤效应(1)普通光照对细胞培养无影响,姜黄素在电镜下能发出土黄色荧光,在灯光照射下对肿瘤细胞原有的杀伤作用增强,荧光显微镜下可见Cur能被肝癌细胞内吞；(2)化学偶联法制备的抗TfR单抗-姜黄素偶联物,其抗体效价较单抗有所降低,但对HepG2肝癌细胞的杀伤作用比姜黄素或抗TfR单抗单独作用时明显增强,与姜黄素+单抗混合物杀伤作用类似；(3)光敏化姜黄素-抗TfR单抗靶向性纳米粒的性质、特征均符合纳米级颗粒：1)薄膜分散法制备的脂质体包封率稳定；2)扫描电镜显示Cur-NP-PEG-Ab形态稳定,属于纳米级；3)FCM分析结果表明Cur-NP-PEG-Ab与鼠源单抗具有相似的结合率；4)FCM分析结果表明在光照条件下Cur-NP-PEG-Ab能影响肝癌细胞的周期S期,并促进其凋亡；5) Cur-NP-PEG-Ab使线粒体电位降低和活性氧产生增加。2.抗TfR抗体基因分析在分泌抗TfR单克隆抗体的同一株杂交瘤细胞中扩增出三条轻链和一条有功能的重链可变区基因,功能性的VH基因片段经DNA凝胶电泳鉴定片段大小与理论值414bp相符,DNA测序显示该基因有完整阅读框架。得到的三条轻链通过IMGT和NCBI分析确认均来自于不同的基因家族：VL-fl引物扩增得到MOPC21骨髓瘤中的非功能内源性轻链;由VL-f2引物扩增出有功能的轻链基因(命名为Vκ2),属于IGKV4-59*0 1家族,可变区长318bp,信号肽长66bp；由VL-f3引物扩增出的功能性轻链基因(命名为Vκ3),属于IGKV6-17*01,可变区长324bp,信号肽长60bp。NCBI数据库报道Vκ3序列与许多单抗轻链可变区相同,也与骨髓瘤MPC11有功能的重排等位基因完全匹配。3D分子模拟显示根据TfR单抗可变区模拟的Fabs结构,两条不同轻链和同一重链偶联制备的Fabs段都具有完整的功能域和结合凹槽。[结论]本研究通过制备可生物降解的mPEG-PLGA-CUR-NPs纳米颗粒,并通过体外实验初步研究了改造的纳米粒生物学作用,获得了有意义的研究结果,研究表明：①制备的Cur-NP-PEG-Ab纳米粒符合纳米级别,具有较高的载药量和包封率；②Cur-NP-PEG-Ab与靶细胞结合率和亲本鼠源性抗体与HepG2细胞结合率相近；③Cur-NP-PEG-Ab在光照下对肿瘤细胞杀伤作用明显增强,提示该纳米粒在完成靶向杀伤肿瘤的同时,能发挥光敏剂的效应；④首次将Cur-NP-PEG-Ab中的姜黄素作为中药光敏剂,脂质体包裹的姜黄素纳米粒,能在体内发挥延长代谢的作用,经过体内循环后,脱去脂质体外衣的姜黄素,又能接着发挥中药本身的抗癌效应。并在此基础上分析了从同一杂交瘤细胞株中扩增出的三条轻链基因,为进一步抗体改造奠定了基础。更多还原

【Abstract】 The project designed to take curcumin as anti-cancer drug and photosensitizer in the light condition. We conjugated modified-curcimin with anti-TfR antibody collaborative to cure cancer. In order to extend drug half-life, to further explore the optical properties of curcumin, we compared different conjugate methods of antibody in combination with the photosensitizer and assessed the coupling efficiency and in vitro testing, discussed the mechanism. Based on the former research, we constructed nano- curcumin and anti-TfR monoclonal antibody conjugate. This work gave experimental evidence for multiple tumor imaging and targeted therapy. Based on analysis of three light chains from the same hybridoma which serecting against TfR mAb, the efforts lay solid foundation for the realization of genetically engineered and genetic engineering targeted anti-tumor effect.[Methods]1. Fabricate and assessment the conjugate of antibody and curcumin.(1) Evaluated tumor cells growth condition in light/non-light by cell count, and determined tumor cell killing effect of photosensitizatied curcumin. Fluorescence microscope to observe HepG2 cells endocytose curcumin.(2) Depended on carbodiimide as the condensing agent, preparated Cur-Ab immunoconjugate chemical, calculated conjugates’antibody titers by indirect ELISA assessed their IC50 by the MTT method.(3) Preparation photosensitizated curcumin-anti-TfR monoclonal antibody actively nanoparticles and preliminary assessment its functions. 1) With a thin film and the injection method to prepare liposomes, and assessed the peak rate of packets.2) Modified curcumin liposomes with TfR mAb.3) Particle morphology studied using scanning electron microscopy.4) FCM detection Cur-NP-PEG-Ab binding rate with cancer cells.5) FCM detection Cur-NP-PEG-Ab in the light and non-light situations on proliferation/apoptosis, cell cycles, etc.6) Used DCFH-DA and ROS kits to assess the changes of mitochondrial potential and ROS.2. Analysis the variable chains of TfR antibody.(1) PCR amplification of VL and VH genes from hybridoma which secertin anti-TfR antibody.(2) Cloning and sequencing of the VH and VL gene by IMGT and NCBI database.(3) Molecular modeling by Insight II to simulate Fabs of against TfR mAb.[Results]1. Fabricated and assessed anti-tumor effect of the conjugate of antibody and curcumin.(1) Normal white light had no effect on cell culture, curcumin can emission khaki-colored fluorescence by electron microscopy, and curcumin killing tumor cells power will be greatly enhanced in the light irradiation, and Curcumin had been endocytosed by HepG2 cells.(2) Coupled of anti-TfR monoclonal antibody and curcumin conjugate in chemical method, the conjugate’s antibody titer decreased. But the killing activity of conjugate super than curcumin, anti-TfR monoclonal antibody on hepatocellular carcinoma cells the killing effect was similar to curcumin+antibody mixture.(3) Active targeted nanoparticles of photosensitizated curcumin conjugate anti-TfR monoclonal antibody characteristics are in line with nano-level:1) The liposome encapsulation has higher stability by film dispersion method. 2) Scanning electron microscopy showed that Cur-NP-PEG-Ab form stable, belonging to nano-level.3) FCM showed that Cur-NP-PEG-Ab has similar binding capacity with the parental murine monoclonal antibody.4) FCM detected under illumination condition, Cur-NP-PEG-Ab can affect hepatoma cells cycle, and promoted their apoptosis.5) Cur-NP-PEG-Ab impacted on mitochondrial potential and ROS, made mitochondrial potential decreased and higher ROS.2. Analysis the variable chains of TfR antibody.Amplified VH and VL genes of anti-TfR monoclonal antibody by PCR, the fragment size in line with the theoretical value, DNA sequencing also showed their are correct sequences. We got the functional light chain different from the former result, belonged to IGKV6-17*01 family has 324bp, and 60bp signal. In order to ensure the accuracy of tests, we tried several amplifications and sequence analyses, confirmed that the light chain was amplified by the primer VL-f3, and the previous sequence in line with VL-f2 primer. So we use a single primer amplification, respectively. We got three different light chain genes from one hybridoma: non-functional endogenous light chain of MOPC21 amplified byVL-fl primers; the primer VL-f2 amplified the variable VL genes was classified to IGKV4-59*01 family has 318bp, and 66bp signal peptide; the primer VL-f3 hybridized the sequence is not the same as in previous one, belonging to different family. Molecular simulation showed the Fabs of the two functional light chains and heavy chain both have binding grooves。[Conclusions]This study was prepared biodegradable mPEG-PLGA-CUR-NPs nanoparticles and preliminary studied the modified nanoparticles biological effects in vitro, obtained meaningful results, studies demonstrated that:(1)Preparation of the Cur-NP-PEG-Ab nanoparticles with high drug loading and encapsulation efficiency complied the nano-level; (2)Cur-NP-PEG-Ab had similar binding rate with target cells (HepG2) to the parental murine antibody; (3)Cur-NP-PEG-Ab enhanced tumor cell killing effect significantly in the light, suggesting that the nanoparticles in the completion of target killing of tumor, can play as a photosensitizer at the same time;(4)the paper first time reported that curcumin in the Cur-NP-PEG-Ab as a herb photosensitizer, liposome-modified-curcumin nanoparticles can play longer in the body metabolism, and after circulated through the body, curcumin coated off liposomes, can then play their anti-cancer effects of Chinese medicine. Based on this analysis of here light chain genes from the same hybridoma, and laid the foundation for the transformation of antibodies.更多还原