新型磁感应热疗介质加热诱导癌细胞凋亡的实验探究及信号肽修饰磁性纳米介质的应用

发布时间：2018-05-18 06:48

本文选题：磁感应热疗 + 细胞凋亡　；参考：《山东大学》2017年硕士论文

【摘要】：实验背景:慢性粒细胞白血病是常见的一种血液系统恶性癌症,导致其愈后效果不好的一大因素是细胞对化疗药物的耐药性。目前,寻求新型治疗技术是该疾病治疗的重点发展方向。微纳介质磁感应热疗技术可应用于血液肿瘤治疗领域,并且能够在体外很好的抑制人白血病细胞的生长,但其诱导凋亡产生的作用机制还不是特别清楚。已知细胞穿膜肽可以携带Fe304纳米粒子进入到细胞中,而线粒体跟细胞核又是细胞中非常重要的两个细胞器,因此我们推测,将磁性纳米粒在线粒体和细胞核富集有利于增强磁感应热疗效果。我们将CPPs穿膜序列与线粒体和细胞核的定位序列相结合,设计出线粒体和细胞核靶向定位的多肽序列,引导磁性纳米粒在细胞内靶向定位。实验目的:1.探讨磁感应热疗技术诱导耐药白血病K562/G细胞凋亡的可能性并探讨其作用机制。2.制备信号肽耦联的磁性纳米粒并在细胞水平探究该纳米粒应用于磁感应热疗的安全性和有效性。实验方法:1.应用耐药白血病K562/G细胞为实验对象,以不锈钢空心球为介质,300kHz,40Gs的磁场条件下43℃热疗30min,流式细胞仪检测细胞的凋亡率,线粒体内膜电位(△ψm)的变化以及细胞周期的阻断情况,探究K562/G死亡是否由凋亡引起;设置43℃,47℃,50℃对细胞进行磁感应热疗,采用比色法检测 Caspase-3,8,9 的活性;Western blotting 分析了 Hsp70 蛋白及β-catenin 蛋白的表达情况;采用免疫荧光法比较Bax/BCl-2比值的变化。2.设计合成RM,RN,TM以及购买鱼精蛋白四种信号肽,共沉淀法合成Fe304纳米粒,在其表面耦联以上四种靶向多肽。采用透射电镜、傅立叶红外光谱仪、Zeta电位分析仪进行物理表观的检测,并绘制磁滞回线。细胞与Omg/mL,8mg/mL,16mg/mL,24mg/mL四种浓度的靶向Fe3O4纳米粒共孵育,24h后MTT法检测合成纳米粒的细胞相容性,使用ICP光谱仪测定共孵育后细胞对纳米粒的吞噬情况,共聚焦显微镜确定粒子的靶向性,最后MTT法检测相同温度下信号肽修饰后的纳米粒子体外升温并抑制细胞增殖的情况。实验结果:1.35、30、25、20、15mg/mL浓度的不锈钢空心球在300kHz、40Gs的磁场下温度升高情况随浓度而增加。MTT法检测空心球24h,48h,72h的细胞毒性较小,细胞相容性较好。细胞在交变磁场下加温30min,43℃细胞的增殖率为73.46±2.60%,47℃细胞的增殖率为66.77±2.34%,5℃细胞的增殖率为55.66±3.24%,磁感应热疗可以很好的杀死以及抑制K562/G细胞的增殖。流式细胞仪检测43℃磁感应热疗30min后,K562/G的凋亡率达到了 39%;线粒体膜电位发生去极化的细胞所占的比例由10.2%增加到了 70.8%;细胞的周期被阻断在G2/M期。磁感应热疗后第24h检测Caspase3,8,9的酶活均显著增强,并且随着加热温度的升高,更多的Caspase3,8,9酶原被激活。经磁热疗后K562/G细胞Hsp70蛋白的表达升高并且随着热疗温度的升高表达增强,而β-catenin蛋白的表达相应降低。免疫荧光检测Bax/Bcl-2的比率随着热疗温度的增加也逐渐增大。2.使用沉淀法反应而成的Fe304,其外形近圆球形,直径大约10nm左右,经信号肽修饰后直径大小和形态均保持良好;MTT法检测其毒性较小,细胞相容性好;Zeta电位有所增加;磁饱和度下降;傅立叶红外光谱仪结果显示耦联后粒子出现了 Fe-O-Fe,Si-O,-CO-NH-收缩震动峰,信号肽成功耦联到了磁流体纳米粒。ICP检测结果表明耦联信号肽后单个细胞中的Fe含量均明显增加。激光共聚焦观察带绿色荧光的Fe304磁性纳米粒在信号肽RM,TM的引导下能够穿过细胞膜并在线粒体部位聚集,而RN-MNPs的靶向性不好。SW480细胞与Fe304磁性纳米粒子在交变磁场下43℃加温30min后,耦联信号肽后的纳米粒子对细胞的热疗效果得到了明显增强,其中PRO-MNPs对细胞的效果最好,细胞的存活率仅为29.54%。实验结论:初步阐明了磁感应热疗技术可以激活细胞的线粒体信号通路,从而导致K562/G细胞发生凋亡。为进一步探究该技术的抑瘤机理打下了良好基础。同时发现信号肽修饰的磁性纳米粒是安全可靠的,在应用于磁感应热疗时,能够显著的提高热疗效率。
[Abstract]:Background: chronic granulocytic leukemia is a common malignant cancer of the blood system. One of the major factors that cause its bad effect is the resistance of cells to chemotherapeutic drugs. At present, seeking new treatment techniques is the key development direction of the disease treatment. Micro nano medium magnetic induction thermotherapy can be used in the field of blood cancer treatment. And it can inhibit the growth of human leukemia cells in vitro, but the mechanism of its induction of apoptosis is not particularly clear. It is known that the membrane peptide can carry Fe304 nanoparticles into the cell, and the mitochondria and the nucleus are two important organelles in the cell. Therefore, we speculate that the magnetic nanoparticles will be magnetic nanoparticles. The accumulation of particles in mitochondria and nuclei helps to enhance the effect of magnetic induction thermotherapy. We combine the CPPs membrane sequence with the localization sequence of mitochondria and nuclei to design the polypeptide sequence of mitochondria and nuclear targeting, and guide the targeting of magnetic nanoparticles in the cell. The purpose of this study is to investigate 1. magnetic induction thermotherapy techniques to induce tolerance. The possibility of apoptosis of leukemic K562/G cells and explore its mechanism of action.2. to prepare magnetic nanoparticles coupled with signal peptide and explore the safety and effectiveness of the nanoparticle applied to magnetic induction thermotherapy at the cell level. Experimental methods: 1. the application of drug resistant leukemia K562/G cells as experimental pairs, stainless steel hollow spheres as medium, 300kHz, 40Gs Under the condition of magnetic field, the apoptosis rate of cell, the change of mitochondrial intima potential (delta m) and the interruption of cell cycle were detected by flow cytometry at 43 C, and the apoptosis caused by K562/G was investigated. The cells were treated by magnetic induction heat therapy at 43, 47 and 50, and Western blot was used to detect the activity of K562/G; Western blot Ting analysis of the expression of Hsp70 protein and beta -catenin protein; using immunofluorescence method to compare the change of Bax/BCl-2 ratio.2. design and synthesis of RM, RN, TM and the purchase of protamine four signal peptides, co precipitation method to synthesize the Fe304 nanoparticles, coupled with the above four target polypeptides. Using transmission electron microscope, Fu Liye infrared spectrometer, Ze. The TA potential analyzer was used to detect the physical appearance and draw the hysteresis loop. The cells were incubated with the target Fe3O4 nanoparticles of four concentrations of Omg/mL, 8mg/mL, 16mg/mL and 24mg/mL. The cell compatibility of the synthesized nanoparticles was detected by MTT method after 24h, and the phagocytosis of the nanoparticles was determined by the ICP spectrometer, and the confocal microscopy was used to determine the particle size. At the same temperature, MTT method was used to detect the temperature of the nanoparticles at the same temperature and inhibit the proliferation of the cells in vitro. The experimental results showed that the temperature of the hollow stainless steel ball with 1.35,30,25,20,15mg/mL concentration increased under the magnetic field of 300kHz and 40Gs with the concentration of.MTT method to detect the cytotoxicity of 24h, 48h, 72h. Small, cell compatibility was better under the alternating magnetic field, the proliferation rate of cells at 43 C was 73.46 + 2.60%, the proliferation rate of cells at 47 C was 66.77 + 2.34%, the proliferation rate of cells at 5 C was 55.66 + 3.24%, magnetic induction thermotherapy could kill well and inhibit the proliferation of K562/G cells. Flow cytometry was used to detect 30min by magnetic induction thermotherapy at 43 C, K The apoptosis rate of 562/G reached 39%, the proportion of the mitochondrial membrane potential depolarizing cells increased from 10.2% to 70.8%, the cell cycle was blocked in the G2/M phase. The enzyme activity of the 24h detection of Caspase3,8,9 after magnetic induction thermotherapy was significantly enhanced, and as the heating temperature increased, more Caspase3,8,9 enzymes were activated. Magnetocaloric The expression of Hsp70 protein in K562/G cells increased and increased with the increase of thermotherapy temperature, and the expression of beta -catenin protein decreased correspondingly. The ratio of immunofluorescence detection Bax/Bcl-2 gradually increased with the precipitation reaction of.2. with the increase of thermotherapy temperature, which was nearly round and around 10nm in diameter. The size and morphology of the modified peptide were good, the MTT method was less toxic and the cell compatibility was better, the Zeta potential was increased and the magnetic saturation decreased. The Fu Liye infrared spectrometer showed that the coupling particles appeared Fe-O-Fe, Si-O, -CO-NH- contraction vibration peak, and the signal peptide was successfully coupled to the magnetic fluid nanoparticles.ICP detection results. The content of Fe in single cells was obviously increased after the coupling signal peptide. The Fe304 magnetic nanoparticles with green fluorescence were observed by confocal laser confocal microscopy, under the guidance of signal peptide RM, TM could cross the cell membrane and gather in the mitochondria, but the target of RN-MNPs was not good for.SW480 and Fe304 magnetic nanoparticles at 43 C under the alternating magnetic field by 3 After 0min, the thermotherapy effect of the nanoparticles after the coupling signal peptide was obviously enhanced, and the effect of PRO-MNPs on the cells was the best. The survival rate of the cells was only the conclusion of the 29.54%. experiment. It was preliminarily clarified that the magnetic induction thermotherapy technology could activate the mitochondrial signal pathway of the cells, thus causing the apoptosis of the K562/G cells. At the same time, it is found that the magnetic nanoparticles modified by the signal peptide is safe and reliable. It can significantly improve the heat treatment efficiency when applied to magnetic induction thermotherapy.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.5

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