本文選題：磁感應(yīng)熱療 + 細(xì)胞凋亡　； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：實(shí)驗(yàn)背景:慢性粒細(xì)胞白血病是常見(jiàn)的一種血液系統(tǒng)惡性癌癥,導(dǎo)致其愈后效果不好的一大因素是細(xì)胞對(duì)化療藥物的耐藥性。目前,尋求新型治療技術(shù)是該疾病治療的重點(diǎn)發(fā)展方向。微納介質(zhì)磁感應(yīng)熱療技術(shù)可應(yīng)用于血液腫瘤治療領(lǐng)域,并且能夠在體外很好的抑制人白血病細(xì)胞的生長(zhǎng),但其誘導(dǎo)凋亡產(chǎn)生的作用機(jī)制還不是特別清楚。已知細(xì)胞穿膜肽可以攜帶Fe304納米粒子進(jìn)入到細(xì)胞中,而線粒體跟細(xì)胞核又是細(xì)胞中非常重要的兩個(gè)細(xì)胞器,因此我們推測(cè),將磁性納米粒在線粒體和細(xì)胞核富集有利于增強(qiáng)磁感應(yīng)熱療效果。我們將CPPs穿膜序列與線粒體和細(xì)胞核的定位序列相結(jié)合,設(shè)計(jì)出線粒體和細(xì)胞核靶向定位的多肽序列,引導(dǎo)磁性納米粒在細(xì)胞內(nèi)靶向定位。實(shí)驗(yàn)?zāi)康?1.探討磁感應(yīng)熱療技術(shù)誘導(dǎo)耐藥白血病K562/G細(xì)胞凋亡的可能性并探討其作用機(jī)制。2.制備信號(hào)肽耦聯(lián)的磁性納米粒并在細(xì)胞水平探究該納米粒應(yīng)用于磁感應(yīng)熱療的安全性和有效性。實(shí)驗(yàn)方法:1.應(yīng)用耐藥白血病K562/G細(xì)胞為實(shí)驗(yàn)對(duì)象,以不銹鋼空心球?yàn)榻橘|(zhì),300kHz,40Gs的磁場(chǎng)條件下43℃熱療30min,流式細(xì)胞儀檢測(cè)細(xì)胞的凋亡率,線粒體內(nèi)膜電位(△ψm)的變化以及細(xì)胞周期的阻斷情況,探究K562/G死亡是否由凋亡引起;設(shè)置43℃,47℃,50℃對(duì)細(xì)胞進(jìn)行磁感應(yīng)熱療,采用比色法檢測(cè) Caspase-3,8,9 的活性;Western blotting 分析了 Hsp70 蛋白及β-catenin 蛋白的表達(dá)情況;采用免疫熒光法比較Bax/BCl-2比值的變化。2.設(shè)計(jì)合成RM,RN,TM以及購(gòu)買魚精蛋白四種信號(hào)肽,共沉淀法合成Fe304納米粒,在其表面耦聯(lián)以上四種靶向多肽。采用透射電鏡、傅立葉紅外光譜儀、Zeta電位分析儀進(jìn)行物理表觀的檢測(cè),并繪制磁滯回線。細(xì)胞與Omg/mL,8mg/mL,16mg/mL,24mg/mL四種濃度的靶向Fe3O4納米粒共孵育,24h后MTT法檢測(cè)合成納米粒的細(xì)胞相容性,使用ICP光譜儀測(cè)定共孵育后細(xì)胞對(duì)納米粒的吞噬情況,共聚焦顯微鏡確定粒子的靶向性,最后MTT法檢測(cè)相同溫度下信號(hào)肽修飾后的納米粒子體外升溫并抑制細(xì)胞增殖的情況。實(shí)驗(yàn)結(jié)果:1.35、30、25、20、15mg/mL濃度的不銹鋼空心球在300kHz、40Gs的磁場(chǎng)下溫度升高情況隨濃度而增加。MTT法檢測(cè)空心球24h,48h,72h的細(xì)胞毒性較小,細(xì)胞相容性較好。細(xì)胞在交變磁場(chǎng)下加溫30min,43℃細(xì)胞的增殖率為73.46±2.60%,47℃細(xì)胞的增殖率為66.77±2.34%,5℃細(xì)胞的增殖率為55.66±3.24%,磁感應(yīng)熱療可以很好的殺死以及抑制K562/G細(xì)胞的增殖。流式細(xì)胞儀檢測(cè)43℃磁感應(yīng)熱療30min后,K562/G的凋亡率達(dá)到了 39%;線粒體膜電位發(fā)生去極化的細(xì)胞所占的比例由10.2%增加到了 70.8%;細(xì)胞的周期被阻斷在G2/M期。磁感應(yīng)熱療后第24h檢測(cè)Caspase3,8,9的酶活均顯著增強(qiáng),并且隨著加熱溫度的升高,更多的Caspase3,8,9酶原被激活。經(jīng)磁熱療后K562/G細(xì)胞Hsp70蛋白的表達(dá)升高并且隨著熱療溫度的升高表達(dá)增強(qiáng),而β-catenin蛋白的表達(dá)相應(yīng)降低。免疫熒光檢測(cè)Bax/Bcl-2的比率隨著熱療溫度的增加也逐漸增大。2.使用沉淀法反應(yīng)而成的Fe304,其外形近圓球形,直徑大約10nm左右,經(jīng)信號(hào)肽修飾后直徑大小和形態(tài)均保持良好;MTT法檢測(cè)其毒性較小,細(xì)胞相容性好;Zeta電位有所增加;磁飽和度下降;傅立葉紅外光譜儀結(jié)果顯示耦聯(lián)后粒子出現(xiàn)了 Fe-O-Fe,Si-O,-CO-NH-收縮震動(dòng)峰,信號(hào)肽成功耦聯(lián)到了磁流體納米粒。ICP檢測(cè)結(jié)果表明耦聯(lián)信號(hào)肽后單個(gè)細(xì)胞中的Fe含量均明顯增加。激光共聚焦觀察帶綠色熒光的Fe304磁性納米粒在信號(hào)肽RM,TM的引導(dǎo)下能夠穿過(guò)細(xì)胞膜并在線粒體部位聚集,而RN-MNPs的靶向性不好。SW480細(xì)胞與Fe304磁性納米粒子在交變磁場(chǎng)下43℃加溫30min后,耦聯(lián)信號(hào)肽后的納米粒子對(duì)細(xì)胞的熱療效果得到了明顯增強(qiáng),其中PRO-MNPs對(duì)細(xì)胞的效果最好,細(xì)胞的存活率僅為29.54%。實(shí)驗(yàn)結(jié)論:初步闡明了磁感應(yīng)熱療技術(shù)可以激活細(xì)胞的線粒體信號(hào)通路,從而導(dǎo)致K562/G細(xì)胞發(fā)生凋亡。為進(jìn)一步探究該技術(shù)的抑瘤機(jī)理打下了良好基礎(chǔ)。同時(shí)發(fā)現(xiàn)信號(hào)肽修飾的磁性納米粒是安全可靠的,在應(yīng)用于磁感應(yīng)熱療時(shí),能夠顯著的提高熱療效率。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.5

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