原子力显微镜研究山奈酚及PD-L1对于淋巴细胞抑制机制

发布时间：2018-06-02 10:56

本文选题：原子力显微镜 + 山奈酚　；参考：《暨南大学》2012年硕士论文

【摘要】：免疫系统是人体对抗疾病，保护正常生理功能的重要系统之一，而疾病的发生、发展与免疫系统对其的反应、变化息息相关。本文分别通过研究程序性凋亡配体1（PD-L1）及山奈酚对Jurkat T细胞的作用，阐释细胞配体及药物在免疫反应中的作用，并得到以下结论： 1，，本文第二章研究了程序性凋亡配体PD-L1对于淋巴细胞的免疫抑制作用。通过CCK-8实验指出程序性凋亡配体对于细胞增殖的抑制效果，该抑制效果随浓度的增加而增强。通过Annexin V/PI细胞凋亡双染试剂盒对细胞凋亡率进行了检测，发现细胞的凋亡率随着配体浓度的增加而从4%增加到73%，进一步佐证了CCK-8实验的结论，指出凋亡配体对于细胞的诱导凋亡作用。实验中还运用流式细胞仪技术分析Jurkat T细胞表面早期活化分子CD69+和共刺激分子PD-1的表达变化情况，CD69+的表达从对照组细胞表面24%下降到实验组细胞的1%，指出细胞的激活状态被明显抑制；而随配体作用浓度的增加，PD-1的表达量也被诱导进一步高表达，从对照组的1.2%增加到实验组的7.3%。通过原子力显微镜对于对照组和实验组的细胞进行分析比较，发现凋亡配体作用后细胞的高度降低、超微结构更加粗糙，进一步解释了细胞功能失活的现象。 2，本文第三章研究了山奈酚对于Jurkat T细胞的诱导凋亡作用。通过增殖抑制实验发现山奈酚对于细胞增殖抑制效果成浓度与时间依赖性，我们通过细胞周期实验发现实验组中处于G2/M期的细胞所占比例随着药物作用浓度的增大也逐渐增大。我们进一步对细胞内Ca~(2+)的表达进行了研究，发现钙离子随着药物作用浓度的增加而从对照组的223(MFI)逐渐增加到实验组的593(MFI)。钙离子的大量释放更加影响了细胞的活性，诱导其加速凋亡。之后通过分子对接实验，模拟了Caspase3，F-actin分别与山奈酚作用的配位情况，指出其潜在的作用位点。最后，通过原子力显微镜对于对照组和实验组的细胞进行分析比较，指出山奈酚作用后细胞的胞体遭到破坏，力学性质等信息产生了明显变化，粘附力和杨氏模量均变小，以上的数据共同解释了细胞功能失活的现象。
[Abstract]:The immune system is one of the important systems for human body to fight diseases and protect normal physiological functions. The occurrence and development of diseases are closely related to the response and changes of the immune system to them. In order to elucidate the role of cell ligands and drugs in immune response, we studied the effects of procedural apoptotic ligand 1 (PD-L 1) and kaempferol on Jurkat T cells, and obtained the following conclusions: 1. In chapter 2, the immunosuppressive effect of programmed apoptotic ligand PD-L1 on lymphocytes was studied. The inhibitory effect of programmed apoptotic ligand on cell proliferation was indicated by CCK-8 assay, and the inhibitory effect increased with the increase of concentration. The apoptosis rate of Annexin V/PI cells was detected by double staining kit. It was found that the apoptosis rate increased from 4% to 73% with the increase of ligand concentration, which further confirmed the conclusion of CCK-8 experiment. The effect of apoptotic ligand on cell apoptosis was pointed out. Flow cytometry was also used to analyze the expression of early activated molecule CD69 and costimulatory molecule PD-1 on the surface of Jurkat T cells. The expression of CD69 decreased from 24% on the surface of the control group to 1 cell in the experimental group. The activation state was obviously inhibited. With the increase of ligand concentration, the expression of PD-1 was further increased from 1.2% in the control group to 7.3% in the experimental group. The results of atomic force microscope (AFM) showed that the cell height decreased and the ultrastructure became rougher after the action of apoptotic ligand, which further explained the phenomenon of cell inactivation. 2. In chapter 3, we studied the apoptosis of Jurkat T cells induced by kaempferol. The inhibitory effect of kaempferol on cell proliferation was found to be concentration-dependent and time-dependent. We found that the proportion of cells in G _ 2 / M phase in experimental group increased with the increase of drug concentration. We further studied the expression of Ca~(2 in cells. It was found that with the increase of the concentration of calcium, calcium gradually increased from 223m MFI in the control group to 593 渭 m MFI in the experimental group. The release of Ca ~ (2 +) significantly affected cell activity and induced apoptosis. The coordination of Caspase3Factin with kaempferol was simulated by molecular docking experiments, and the potential sites of Caspase3 F-actin were pointed out. Finally, the cells of the control group and experimental group were analyzed and compared by atomic force microscope. It was pointed out that the cell body was destroyed, the mechanical properties of the cells were obviously changed, and the adhesion force and Young's modulus were both decreased after the treatment of kaempferol. The above data together explain the inactivation of cell function.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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