抗膀胱癌抗體BCMab1與順鉑協(xié)同抗膀胱癌的拉曼光譜研究
發(fā)布時(shí)間：2018-09-11 19:01
【摘要】：[背景和目的]膀胱癌是我過最常見的泌尿生殖系惡性腫瘤,因其有較高的復(fù)發(fā)率,并且每次復(fù)發(fā)其分期分級有進(jìn)展可能,膀胱癌的復(fù)發(fā)防治技術(shù)一直以來是學(xué)術(shù)界關(guān)注的重要課題。膀胱癌復(fù)發(fā)的主要原因是手術(shù)不能完全切除所有的腫瘤細(xì)胞,因此需探索新的治療方案,既能夠減少膀胱癌術(shù)后復(fù)發(fā)的幾率,又能減少耐藥和減輕藥物的副作用。在膀胱癌的治療方法中,腫瘤的免疫治療通過激發(fā)或聯(lián)合機(jī)體的免疫系統(tǒng),強(qiáng)化機(jī)體對腫瘤免疫力,同時(shí)介導(dǎo)免疫系統(tǒng)對腫瘤細(xì)胞進(jìn)行殺滅清除。激光拉曼光譜作為一種簡單、快捷、無損、高靈敏的檢測方法通過光譜學(xué)檢測能夠?qū)ξ镔|(zhì)分子結(jié)構(gòu)的特異性改變進(jìn)行分析。組織或細(xì)胞的拉曼光譜主要分析其相關(guān)成分如糖類、蛋白、脂類、核酸,和其內(nèi)部組分小分子的內(nèi)部化學(xué)鍵振動變化。當(dāng)細(xì)胞內(nèi)部成分的含量、結(jié)構(gòu)改變時(shí)就有可能形成特征的拉曼峰。我們采用膀胱癌特異性抗體(BCMab1)聯(lián)合順鉑進(jìn)行體外細(xì)胞研究,通過拉曼光譜對分子特征構(gòu)象改變進(jìn)行檢測,分析經(jīng)過不同方法處理后的細(xì)胞特征拉曼峰的改變,推斷膀胱癌特異性抗體(BCMab1)與順鉑之間是否存在協(xié)同抗膀胱癌效果,探索膀胱癌特異性抗體(BCMab1)和順鉑作用膀胱癌細(xì)胞后的內(nèi)部分子改變機(jī)制,為抗腫瘤藥物聯(lián)合治療提供研究基礎(chǔ)。[材料與方法]膀胱癌EJ細(xì)胞系,用于制備抗膀胱癌抗體BCMab1所用的雜交瘤細(xì)胞,制作腹水的Balb/c小鼠,順鉑均由中國科學(xué)院生物物理研究所提供(一)首先采用腹水發(fā)制備單克隆抗體,然后對進(jìn)行粗球制備抗體純化采用流式細(xì)胞儀對抗體檢驗(yàn)。(二)取對數(shù)生長期的膀胱癌細(xì)胞系EJ細(xì)胞采用蓋玻片加六孔板按照設(shè)計(jì)方案進(jìn)行培養(yǎng)：對照組(不加藥物處理)單獨(dú)抗組(BCMab1終濃度為25 μg/ml和50μg/ml)單獨(dú)順鉑組(DDP終濃度25 μ g/ml和50 μ g/ml)抗體與順鉑混合組(終濃度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)進(jìn)行培養(yǎng)48h后(每個組設(shè)置3個重復(fù))用多聚甲醛固定細(xì)胞,梯度濃度乙醇進(jìn)行脫去固定液和細(xì)胞內(nèi)水分,固定細(xì)胞狀態(tài)后采用785nm激發(fā)光拉曼光譜儀進(jìn)行拉曼光譜采集,并參考文獻(xiàn)總結(jié)拉曼譜帶歸屬,通過對特征峰位征意義進(jìn)行分析,探索膀胱癌細(xì)胞EJ系的分子生化改變,所得數(shù)據(jù)用origin8.6專業(yè)版進(jìn)行行數(shù)據(jù)和圖譜處理[實(shí)驗(yàn)結(jié)果]1.不加任何藥物對照組,單獨(dú)加抗體組,單獨(dú)加順鉑組,加抗體和順鉑混合藥物組,在培養(yǎng)48h的平均拉曼光譜均在1450 cm-1,1170 cm-1,1031 cm-1,1260 cm-1,1094 cm-1,1209 cm-1表現(xiàn)出特征拉曼峰；2.單獨(dú)順鉑的整體平均拉曼峰位曲線均明顯低于單獨(dú)加順鉑的曲線且濃度越高越明顯；3.單獨(dú)加抗體的整體拉曼曲線均低于陰性對照組平均拉曼峰位曲線但不明顯；4.單獨(dú)加入抗體組,單獨(dú)加入順鉑組,抗體和順鉑混合組,并沒有出現(xiàn)明顯的新的特征的拉曼峰[結(jié)論]1.作為細(xì)胞周期非特異性藥物順鉑對細(xì)胞凋亡作用的效果可以用拉曼光譜進(jìn)行整體性評價(jià),但具體的作用靶點(diǎn)以及詳細(xì)的作用效果不能分辨出。且其本身作用膀胱癌EJ細(xì)胞后不能產(chǎn)生其獨(dú)有的拉曼特異性位移峰的出現(xiàn)。2.抗膀胱癌抗體(BCMab1)對細(xì)胞凋亡作用的效果可以用拉曼光譜進(jìn)行整體性評價(jià),但具體的作為靶點(diǎn)以及詳細(xì)的作用效果不能分辨出。且其本身作用膀胱癌EJ細(xì)胞后不能產(chǎn)生其獨(dú)有的拉曼特異性位移峰的出現(xiàn)。3.抗膀胱癌抗體(BCMab1)和順鉑(DDP)聯(lián)合作用于EJ細(xì)胞后產(chǎn)生的效果可以用拉曼光譜進(jìn)行整體性評價(jià),但具體的作用靶點(diǎn)以及詳細(xì)的作用效果不能分辨出。且聯(lián)合作用膀胱癌EJ細(xì)胞后不能產(chǎn)生其獨(dú)有的拉曼特異性位移峰的出現(xiàn)。4.參考以往通過組織拉曼膀胱研究的結(jié)論發(fā)現(xiàn)膀胱癌EJ細(xì)胞系同樣具有部分組織拉曼特異性的位移峰。5.通過本實(shí)驗(yàn)?zāi)軌蜃C明抗膀胱癌抗體(BCMab1)和順鉑(DDP)聯(lián)合作用于EJ細(xì)胞不具有明顯的協(xié)同效應(yīng)。
[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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R737.14;O657.37
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