尼古丁致人脐带间充质干细胞凋亡及蛋白差异表达的研究

发布时间：2018-04-10 14:39

  本文选题：尼古丁 + 间充质干细胞　； 参考：《暨南大学》2012年硕士论文

【摘要】：目的：探讨尼古丁对人脐带间充质干细胞（human umbilical cord mesenchymal stem cells,hMSCs）表面形貌、增殖、凋亡等指标的影响，并研究其分子信号通路和蛋白质表达谱的变化，寻找尼古丁作用MSCs的靶点，为MSCs治疗吸烟患者及吸烟相关性疾病提供实验依据。 方法：(1)用相差显微镜及原子力显微镜观察尼古丁作用前后人脐带MSCs细胞表面形貌的变化；(2)不同浓度尼古丁(0.5mg/mL,1.0mg/mL,1.5mg/mL)作用于人脐带MSCs，于24h、48h、72h用MTT法检测细胞增殖活性；(3)流式细胞仪检测尼古丁对人脐带MSCs细胞凋亡、细胞周期的影响；(4)流式细胞仪检测尼古丁作用后人脐带MSCs活性氧、线粒体膜电位、细胞内钙离子的变化；(5)用硝酸还原酶法检测不同浓度尼古丁作用MSCs后24h、36h、48h，细胞内一氧化氮（NO）的释放情况；(6)实时荧光定量PCR检测尼古丁作用后人脐带MSCs内α7nAchR的表达情况；(7)双向凝胶电泳技术（2-DE）分离尼古丁作用前后的人脐带MSCs总蛋白，ImageMaster2D Platinum软件分析蛋白质差异表达点，基质辅助激光解析串联飞行时间质谱对差异表达的蛋白进行鉴定及功能分类。 结果：(1)正常MSCs呈长梭形、多角形等不规则形态，细胞生长较快，呈融合生长。尼古丁作用后，细胞固缩，细胞膜受到破坏，胞质中产生大量空泡，，胞核膨胀突起；(2)尼古丁抑制人脐带MSCs生长，呈时间剂量依赖性；(3)尼古丁作用后，细胞周期分布发生改变，G0／G1期细胞明显增加，G2期和S期则逐渐减少，同时出现较高的凋亡率，周期阻滞及凋亡率随浓度递增而增加；(4)尼古丁作用MSCs后，细胞内钙离子及活性氧升高，细胞线粒体膜电位下降；(5)尼古丁作用MSCs24h、36h后，各实验组NO水平显著高于对照组（P0.05），呈时间、浓度依赖性，但在48h，0.8mg/ml与1.0mg/ml组，NO水平低于对照组；(6)人脐带MSCs表达α7nAchR，尼古丁作用细胞后表达升高，呈时间浓度依赖性；(7)尼古丁作用人脐带MSCs后，蛋白质谱表达发生改变，鉴定出27个差异表达蛋白，其中13个表达上调，14个表达下调。 结论：尼古丁对人脐带MSCs有毒性作用，可使细胞表面形貌发生改变；并抑制细胞增殖、促进其凋亡。尼古丁诱导人脐带MSCs凋亡涉及多信号及多蛋白通路，它可通过激活nAchR下游通路，升高细胞内钙离子、活性氧、一氧化氮水平，降低MSCs线粒体膜电位促进细胞凋亡。尼古丁使MSCs蛋白质谱表达发生改变，差异蛋白功能涉及细胞骨架结构和运动、信号转导、离子通道蛋白、细胞代谢、肌肉收缩等。
[Abstract]:Objective: to investigate the effects of nicotine on the surface morphology, proliferation and apoptosis of human umbilical cord mesenchymal stem cells in human umbilical cord mesenchymal stem cells (hMSCs), and to study the changes of molecular signal pathway and protein expression profile in order to find the target of nicotine acting on MSCs.To provide experimental evidence for the treatment of smoking patients and smoking related diseases by MSCs.Apoptotic effect of nicotine on human umbilical cord MSCs cells was detected by cytometer.Effects of nicotine on cell cycle: flow cytometry was used to detect reactive oxygen species (Ros) and mitochondrial membrane potential (MMP) in human umbilical cord MSCs after nicotine treatment.The changes of intracellular Ca ~ (2 +) I ~ (5) the release of nitric oxide (no) in human umbilical cord MSCs was detected by nitric acid reductase method at 24 h, 36 h and 48 h after exposure to nicotine. The expression of 伪 7nAchR in human umbilical cord MSCs was detected by real-time fluorescence quantitative PCR.Two-dimensional gel electrophoresis (2-DEE) was used to analyze protein differential expression points in human umbilical cord MSCs total protein before and after nicotine treatment by ImageMaster2D Platinum software.Matrix assisted laser desorption tandem time of flight mass spectrometry was used to identify and classify differentially expressed proteins.Results (1) normal MSCs showed long spindle shape, polygonal shape and irregular shape. The cells grew faster and showed fusion growth.After nicotine treatment, the cells became pyknotic, the cell membrane was destroyed, a large number of vacuoles were produced in the cytoplasm, and the nuclear swelling process was 2) nicotine inhibited the growth of MSCs in human umbilical cord in a time and dose-dependent manner.Cell cycle distribution was changed. Cell cycle arrest and apoptosis rate increased with increasing concentration in G _ 0 / G _ 1 phase, but decreased gradually in G _ 2 phase and S phase.The levels of no in each experimental group were significantly higher than those in the control group (P 0.05) in a time-and concentration-dependent manner after treatment of MSCs for 24 h or 36 h with the increase of intracellular calcium ion and reactive oxygen species, and the decrease of mitochondrial membrane potential of the cells was found in a time-dependent and concentration-dependent manner.The expression of 伪 7nAchRin MSCs in human umbilical cord was significantly lower than that in control group (P < 0.05), but the expression of 伪 7nAchRin in human umbilical cord MSCs was increased after treatment with nicotine in a dose-dependent manner (P < 0.05).27 differentially expressed proteins were identified, of which 13 were up-regulated and 14 down-regulated.Conclusion: nicotine has toxic effect on human umbilical cord MSCs, which can change cell surface morphology, inhibit cell proliferation and promote apoptosis.Nicotine induces apoptosis of human umbilical cord MSCs involved in multiple signal and polyprotein pathways, which can promote apoptosis by activating downstream nAchR pathway, increasing intracellular calcium ion, reactive oxygen species and nitric oxide levels, and decreasing mitochondrial membrane potential of MSCs.Nicotine changes the expression of MSCs protein profile. The function of differential protein involves cytoskeleton structure and movement, signal transduction, ion channel protein, cell metabolism, muscle contraction and so on.
【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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