抗膀胱癌抗体BCMab1与顺铂协同抗膀胱癌的拉曼光谱研究
发布时间：2018-09-11 19:01
【摘要】：[背景和目的]膀胱癌是我过最常见的泌尿生殖系恶性肿瘤,因其有较高的复发率,并且每次复发其分期分级有进展可能,膀胱癌的复发防治技术一直以来是学术界关注的重要课题。膀胱癌复发的主要原因是手术不能完全切除所有的肿瘤细胞,因此需探索新的治疗方案,既能够减少膀胱癌术后复发的几率,又能减少耐药和减轻药物的副作用。在膀胱癌的治疗方法中,肿瘤的免疫治疗通过激发或联合机体的免疫系统,强化机体对肿瘤免疫力,同时介导免疫系统对肿瘤细胞进行杀灭清除。激光拉曼光谱作为一种简单、快捷、无损、高灵敏的检测方法通过光谱学检测能够对物质分子结构的特异性改变进行分析。组织或细胞的拉曼光谱主要分析其相关成分如糖类、蛋白、脂类、核酸,和其内部组分小分子的内部化学键振动变化。当细胞内部成分的含量、结构改变时就有可能形成特征的拉曼峰。我们采用膀胱癌特异性抗体(BCMab1)联合顺铂进行体外细胞研究,通过拉曼光谱对分子特征构象改变进行检测,分析经过不同方法处理后的细胞特征拉曼峰的改变,推断膀胱癌特异性抗体(BCMab1)与顺铂之间是否存在协同抗膀胱癌效果,探索膀胱癌特异性抗体(BCMab1)和顺铂作用膀胱癌细胞后的内部分子改变机制,为抗肿瘤药物联合治疗提供研究基础。[材料与方法]膀胱癌EJ细胞系,用于制备抗膀胱癌抗体BCMab1所用的杂交瘤细胞,制作腹水的Balb/c小鼠,顺铂均由中国科学院生物物理研究所提供(一)首先采用腹水发制备单克隆抗体,然后对进行粗球制备抗体纯化采用流式细胞仪对抗体检验。(二)取对数生长期的膀胱癌细胞系EJ细胞采用盖玻片加六孔板按照设计方案进行培养：对照组(不加药物处理)单独抗组(BCMab1终浓度为25 μg/ml和50μg/ml)单独顺铂组(DDP终浓度25 μ g/ml和50 μ g/ml)抗体与顺铂混合组(终浓度BCMab1,25μ g/ml+DDP,25μ g/ml; BCMab1,25μ g/ml+DDP,50μ g/ml; BCMab1,50 μ g/ml+DDP,25 μ g/ml; BCMab1,50 μ g/ml+DDP,25 μ g/ml)进行培养48h后(每个组设置3个重复)用多聚甲醛固定细胞,梯度浓度乙醇进行脱去固定液和细胞内水分,固定细胞状态后采用785nm激发光拉曼光谱仪进行拉曼光谱采集,并参考文献总结拉曼谱带归属,通过对特征峰位征意义进行分析,探索膀胱癌细胞EJ系的分子生化改变,所得数据用origin8.6专业版进行行数据和图谱处理[实验结果]1.不加任何药物对照组,单独加抗体组,单独加顺铂组,加抗体和顺铂混合药物组,在培养48h的平均拉曼光谱均在1450 cm-1,1170 cm-1,1031 cm-1,1260 cm-1,1094 cm-1,1209 cm-1表现出特征拉曼峰；2.单独顺铂的整体平均拉曼峰位曲线均明显低于单独加顺铂的曲线且浓度越高越明显；3.单独加抗体的整体拉曼曲线均低于阴性对照组平均拉曼峰位曲线但不明显；4.单独加入抗体组,单独加入顺铂组,抗体和顺铂混合组,并没有出现明显的新的特征的拉曼峰[结论]1.作为细胞周期非特异性药物顺铂对细胞凋亡作用的效果可以用拉曼光谱进行整体性评价,但具体的作用靶点以及详细的作用效果不能分辨出。且其本身作用膀胱癌EJ细胞后不能产生其独有的拉曼特异性位移峰的出现。2.抗膀胱癌抗体(BCMab1)对细胞凋亡作用的效果可以用拉曼光谱进行整体性评价,但具体的作为靶点以及详细的作用效果不能分辨出。且其本身作用膀胱癌EJ细胞后不能产生其独有的拉曼特异性位移峰的出现。3.抗膀胱癌抗体(BCMab1)和顺铂(DDP)联合作用于EJ细胞后产生的效果可以用拉曼光谱进行整体性评价,但具体的作用靶点以及详细的作用效果不能分辨出。且联合作用膀胱癌EJ细胞后不能产生其独有的拉曼特异性位移峰的出现。4.参考以往通过组织拉曼膀胱研究的结论发现膀胱癌EJ细胞系同样具有部分组织拉曼特异性的位移峰。5.通过本实验能够证明抗膀胱癌抗体(BCMab1)和顺铂(DDP)联合作用于EJ细胞不具有明显的协同效应。
[Abstract]:[BACKGROUND AND OBJECTIVE] Bladder cancer is the most common malignant tumor of urogenital system in my life. Because of its high recurrence rate and the possibility of progression in staging and grading of each recurrence, the prevention and treatment of recurrence of bladder cancer has always been an important topic of academic concern. In the treatment of bladder cancer, tumor immunotherapy enhances the immunity of the body to the tumor by stimulating or combining the immune system of the body and mediates the immune system to the tumor. Laser Raman spectroscopy is a simple, fast, nondestructive and highly sensitive method for the analysis of specific changes in the molecular structure of substances. Raman spectroscopy of tissues or cells mainly analyzes the related components such as carbohydrates, proteins, lipids, nucleic acids, and small molecules of their internal components. We used BCMab1 in combination with cisplatin to study the cell in vitro. Raman spectroscopy was used to detect the changes of molecular characteristic conformation and analyze the cell characteristic Raman spectra after different treatment methods. The changes of Mann peak could be used to infer whether there is synergistic effect between BCMab1 and cisplatin on bladder cancer, and to explore the mechanism of intracellular molecular changes after BCMab1 and cisplatin acting on bladder cancer cells, so as to provide a basis for the study of combined antineoplastic therapy. [Materials and Methods] Bladder cancer EJ cell line was used for bladder cancer treatment. Hybridoma cells used to prepare anti-bladder cancer antibody BCMab1 were used to prepare Balb/c mice with ascites. Cisplatin was provided by Institute of Biophysics, Chinese Academy of Sciences. (1) Monoclonal antibodies were prepared from ascites, then crude antibodies were purified and tested by flow cytometry. Cell line EJ cells were cultured with covered slides and six-hole plate according to the design: control group (without drug treatment) alone anti-group (BCMab1 final concentration 25 ug/ml and 50 ug/ml) alone cisplatin group (DDP final concentration 25 ug/ml and 50 ug/ml) antibody mixed with cisplatin group (final concentration BCMab1,25 ug/ml+DDP, 25 ug/ml; G/ml; BCMab 1,50 ug/ml + DDP, 25 ug/ml; BCMab 1,50 ug/ml + DDP, 25 ug/ml) were cultured for 48 hours (each group set up three replicates) with paraformaldehyde to fix the cells, and ethanol with gradient concentration was used to remove the stationary solution and intracellular water. After fixing the cells, the Raman spectra were collected by 785 nm excitation light Raman spectrometer, and references were made. Raman band assignment was summarized. The molecular biochemical changes of EJ cell line of bladder cancer were explored by analyzing the significances of characteristic peaks. The data were processed with the professional version of origin 8.6. The average Raman spectra at 48h showed characteristic Raman peaks at 1450 cm-1,1170 cm-1,1031 cm-1,1260 cm-1,1094 cm-1,1209 cm-1. The average Raman peak curve was not obvious in group A. The effect of cisplatin on cell apoptosis could be evaluated by Raman spectroscopy, but the effect of cisplatin on cell apoptosis could be evaluated by Raman spectroscopy. The specific Raman shift peaks could not be produced after the action of BCMab1 on bladder cancer EJ cells. 2. The effect of anti-bladder cancer antibody (BCMab1) on cell apoptosis can be evaluated by Raman spectroscopy as a whole, but as a specific target and a detailed role. The effect can not be distinguished. Moreover, it can not produce its own Raman-specific shift peak after it acts on bladder cancer EJ cells. 3. The combined effect of anti-bladder cancer antibody (BCMab1) and cisplatin (DDP) on EJ cells can be evaluated by Raman spectroscopy as a whole, but the specific target and detailed effect are not. It can be distinguished. And the combination of EJ cells of bladder cancer can not produce its unique Raman-specific shift peak. 4. Referring to the previous results of tissue Raman bladder studies, we found that bladder cancer EJ cell lines also have some tissue Raman-specific shift peaks. 5. This experiment can prove that anti-bladder cancer antibody (BCMab1) and cis. Platinum (DDP) has no synergistic effect on EJ cells.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.14;O657.37
[Abstract]:[BACKGROUND AND OBJECTIVE] Bladder cancer is the most common malignant tumor of urogenital system in my life. Because of its high recurrence rate and the possibility of progression in staging and grading of each recurrence, the prevention and treatment of recurrence of bladder cancer has always been an important topic of academic concern. In the treatment of bladder cancer, tumor immunotherapy enhances the immunity of the body to the tumor by stimulating or combining the immune system of the body and mediates the immune system to the tumor. Laser Raman spectroscopy is a simple, fast, nondestructive and highly sensitive method for the analysis of specific changes in the molecular structure of substances. Raman spectroscopy of tissues or cells mainly analyzes the related components such as carbohydrates, proteins, lipids, nucleic acids, and small molecules of their internal components. We used BCMab1 in combination with cisplatin to study the cell in vitro. Raman spectroscopy was used to detect the changes of molecular characteristic conformation and analyze the cell characteristic Raman spectra after different treatment methods. The changes of Mann peak could be used to infer whether there is synergistic effect between BCMab1 and cisplatin on bladder cancer, and to explore the mechanism of intracellular molecular changes after BCMab1 and cisplatin acting on bladder cancer cells, so as to provide a basis for the study of combined antineoplastic therapy. [Materials and Methods] Bladder cancer EJ cell line was used for bladder cancer treatment. Hybridoma cells used to prepare anti-bladder cancer antibody BCMab1 were used to prepare Balb/c mice with ascites. Cisplatin was provided by Institute of Biophysics, Chinese Academy of Sciences. (1) Monoclonal antibodies were prepared from ascites, then crude antibodies were purified and tested by flow cytometry. Cell line EJ cells were cultured with covered slides and six-hole plate according to the design: control group (without drug treatment) alone anti-group (BCMab1 final concentration 25 ug/ml and 50 ug/ml) alone cisplatin group (DDP final concentration 25 ug/ml and 50 ug/ml) antibody mixed with cisplatin group (final concentration BCMab1,25 ug/ml+DDP, 25 ug/ml; G/ml; BCMab 1,50 ug/ml + DDP, 25 ug/ml; BCMab 1,50 ug/ml + DDP, 25 ug/ml) were cultured for 48 hours (each group set up three replicates) with paraformaldehyde to fix the cells, and ethanol with gradient concentration was used to remove the stationary solution and intracellular water. After fixing the cells, the Raman spectra were collected by 785 nm excitation light Raman spectrometer, and references were made. Raman band assignment was summarized. The molecular biochemical changes of EJ cell line of bladder cancer were explored by analyzing the significances of characteristic peaks. The data were processed with the professional version of origin 8.6. The average Raman spectra at 48h showed characteristic Raman peaks at 1450 cm-1,1170 cm-1,1031 cm-1,1260 cm-1,1094 cm-1,1209 cm-1. The average Raman peak curve was not obvious in group A. The effect of cisplatin on cell apoptosis could be evaluated by Raman spectroscopy, but the effect of cisplatin on cell apoptosis could be evaluated by Raman spectroscopy. The specific Raman shift peaks could not be produced after the action of BCMab1 on bladder cancer EJ cells. 2. The effect of anti-bladder cancer antibody (BCMab1) on cell apoptosis can be evaluated by Raman spectroscopy as a whole, but as a specific target and a detailed role. The effect can not be distinguished. Moreover, it can not produce its own Raman-specific shift peak after it acts on bladder cancer EJ cells. 3. The combined effect of anti-bladder cancer antibody (BCMab1) and cisplatin (DDP) on EJ cells can be evaluated by Raman spectroscopy as a whole, but the specific target and detailed effect are not. It can be distinguished. And the combination of EJ cells of bladder cancer can not produce its unique Raman-specific shift peak. 4. Referring to the previous results of tissue Raman bladder studies, we found that bladder cancer EJ cell lines also have some tissue Raman-specific shift peaks. 5. This experiment can prove that anti-bladder cancer antibody (BCMab1) and cis. Platinum (DDP) has no synergistic effect on EJ cells.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.14;O657.37
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