發(fā)布時(shí)間：2018-04-10 14:39

  本文選題：尼古丁 + 間充質(zhì)干細(xì)胞��； 參考：《暨南大學(xué)》2012年碩士論文

【摘要】：目的：探討尼古丁對(duì)人臍帶間充質(zhì)干細(xì)胞（human umbilical cord mesenchymal stem cells,hMSCs）表面形貌、增殖、凋亡等指標(biāo)的影響，并研究其分子信號(hào)通路和蛋白質(zhì)表達(dá)譜的變化，尋找尼古丁作用MSCs的靶點(diǎn)，為MSCs治療吸煙患者及吸煙相關(guān)性疾病提供實(shí)驗(yàn)依據(jù)。 方法：(1)用相差顯微鏡及原子力顯微鏡觀察尼古丁作用前后人臍帶MSCs細(xì)胞表面形貌的變化；(2)不同濃度尼古丁(0.5mg/mL,1.0mg/mL,1.5mg/mL)作用于人臍帶MSCs，于24h、48h、72h用MTT法檢測(cè)細(xì)胞增殖活性；(3)流式細(xì)胞儀檢測(cè)尼古丁對(duì)人臍帶MSCs細(xì)胞凋亡、細(xì)胞周期的影響；(4)流式細(xì)胞儀檢測(cè)尼古丁作用后人臍帶MSCs活性氧、線粒體膜電位、細(xì)胞內(nèi)鈣離子的變化；(5)用硝酸還原酶法檢測(cè)不同濃度尼古丁作用MSCs后24h、36h、48h，細(xì)胞內(nèi)一氧化氮（NO）的釋放情況；(6)實(shí)時(shí)熒光定量PCR檢測(cè)尼古丁作用后人臍帶MSCs內(nèi)α7nAchR的表達(dá)情況；(7)雙向凝膠電泳技術(shù)（2-DE）分離尼古丁作用前后的人臍帶MSCs總蛋白，ImageMaster2D Platinum軟件分析蛋白質(zhì)差異表達(dá)點(diǎn)，基質(zhì)輔助激光解析串聯(lián)飛行時(shí)間質(zhì)譜對(duì)差異表達(dá)的蛋白進(jìn)行鑒定及功能分類。 結(jié)果：(1)正常MSCs呈長(zhǎng)梭形、多角形等不規(guī)則形態(tài)，細(xì)胞生長(zhǎng)較快，呈融合生長(zhǎng)。尼古丁作用后，細(xì)胞固縮，細(xì)胞膜受到破壞，胞質(zhì)中產(chǎn)生大量空泡，，胞核膨脹突起；(2)尼古丁抑制人臍帶MSCs生長(zhǎng)，呈時(shí)間劑量依賴性；(3)尼古丁作用后，細(xì)胞周期分布發(fā)生改變，G0／G1期細(xì)胞明顯增加，G2期和S期則逐漸減少，同時(shí)出現(xiàn)較高的凋亡率，周期阻滯及凋亡率隨濃度遞增而增加；(4)尼古丁作用MSCs后，細(xì)胞內(nèi)鈣離子及活性氧升高，細(xì)胞線粒體膜電位下降；(5)尼古丁作用MSCs24h、36h后，各實(shí)驗(yàn)組NO水平顯著高于對(duì)照組（P0.05），呈時(shí)間、濃度依賴性，但在48h，0.8mg/ml與1.0mg/ml組，NO水平低于對(duì)照組；(6)人臍帶MSCs表達(dá)α7nAchR，尼古丁作用細(xì)胞后表達(dá)升高，呈時(shí)間濃度依賴性；(7)尼古丁作用人臍帶MSCs后，蛋白質(zhì)譜表達(dá)發(fā)生改變，鑒定出27個(gè)差異表達(dá)蛋白，其中13個(gè)表達(dá)上調(diào)，14個(gè)表達(dá)下調(diào)。 結(jié)論：尼古丁對(duì)人臍帶MSCs有毒性作用，可使細(xì)胞表面形貌發(fā)生改變；并抑制細(xì)胞增殖、促進(jìn)其凋亡。尼古丁誘導(dǎo)人臍帶MSCs凋亡涉及多信號(hào)及多蛋白通路，它可通過(guò)激活nAchR下游通路，升高細(xì)胞內(nèi)鈣離子、活性氧、一氧化氮水平，降低MSCs線粒體膜電位促進(jìn)細(xì)胞凋亡。尼古丁使MSCs蛋白質(zhì)譜表達(dá)發(fā)生改變，差異蛋白功能涉及細(xì)胞骨架結(jié)構(gòu)和運(yùn)動(dòng)、信號(hào)轉(zhuǎn)導(dǎo)、離子通道蛋白、細(xì)胞代謝、肌肉收縮等。
[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 偽 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 偽 7nAchRin MSCs in human umbilical cord was significantly lower than that in control group (P < 0.05), but the expression of 偽 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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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