本文選題：納米磁流體 + 磁流體熱療��； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：口腔鱗狀細(xì)胞癌(oral squamous cell carcinoma, OSSC)是口腔頜面部最常見的惡性腫瘤,來源于皮膚和黏膜的復(fù)層鱗狀上皮細(xì)胞。而在口腔鱗狀細(xì)胞癌中,最常見的類型之一就是舌鱗狀細(xì)胞癌。近年來口腔鱗狀細(xì)胞癌的發(fā)病率呈上升趨勢,發(fā)病年齡也趨于年輕化。尋找積極有效的治療途徑成為一項(xiàng)十分迫切的重要任務(wù)。手術(shù)療法、化學(xué)療法、放射療法依然是目前最主要的三種治療舌鱗狀細(xì)胞癌的手段,但手術(shù)治療的損傷和風(fēng)險(xiǎn)以及放化療的巨大毒副作用嚴(yán)重影響了腫瘤患者的生存質(zhì)量和存活率。因此,研究和開發(fā)具有靶向性的、更加安全、更加有效、低副作用的、無創(chuàng)或微創(chuàng)的舌鱗狀細(xì)胞癌治療新方案勢在必行。 利用腫瘤組織較正常組織對高溫更加敏感的原理,將腫瘤組織加熱到43℃以上,腫瘤細(xì)胞較正常細(xì)胞不能耐受持續(xù)高溫,從而發(fā)生不同程度的凋亡或壞死,這種治療惡性腫瘤的方法稱熱療(Hyperthermia)。近年來,納米技術(shù)的迅速發(fā)展使其成為在醫(yī)學(xué)領(lǐng)域具有挑戰(zhàn)性的創(chuàng)新。磁流體熱療(Magnetic fluid hyperthermia, MFH)是將經(jīng)過包裹修飾或添加了某種抗體的磁流體通過動(dòng)靜脈或直接注射到達(dá)腫瘤內(nèi)部,磁流體被腫瘤細(xì)胞吞噬或沉積在細(xì)胞之間,在交變磁場(Alternating magnetic field, AMF)下,磁性納米顆粒通過磁矢量旋轉(zhuǎn)和顆粒本身的物理旋轉(zhuǎn),即通過奈爾松弛和布朗弛豫機(jī)制升溫,從而殺死腫瘤細(xì)胞,而周圍正常組織升溫不明顯,具有高度的靶向性。磁流體熱療技術(shù)的出現(xiàn)解決了熱療中病灶準(zhǔn)確定位的問題,在腫瘤熱療方面取得較好的療效。磁流體熱療是一項(xiàng)對深部組織熱療的全新方法,可以通過調(diào)節(jié)磁流體的濃度或磁場強(qiáng)度來得到我們實(shí)驗(yàn)中想要的熱療溫度,本實(shí)驗(yàn)是通過調(diào)節(jié)磁流體濃度來實(shí)現(xiàn)的；局部熱療法可分為溫?zé)岑煼?42-46℃)、高熱療法(46-70℃)、熱切除(70℃以上)。與傳統(tǒng)的熱療技術(shù)如紅外熱療、超聲熱療、微波熱療和射頻消融、電磁波熱療相比,MFH技術(shù)具有可治療深層腫瘤、副作用小、微創(chuàng)甚至無創(chuàng)的特點(diǎn),且靶向性好,且腫瘤部位單次注射納米磁流體,可以對病人進(jìn)行重復(fù)多次熱療。 裸鼠免疫功能缺陷,在一定條件下,不排斥來自異種動(dòng)物的組織移植；被移植腫瘤仍保持原有組織學(xué)結(jié)構(gòu)和生物學(xué)特性；同時(shí),幾乎很少見移植腫瘤發(fā)生轉(zhuǎn)移。這些條件是成功建立人舌癌腫瘤模型的基礎(chǔ),也是本實(shí)驗(yàn)的重要保證；且裸鼠腋窩皮下腫瘤模型,腫瘤組織生長表淺易從體外捫及,腫瘤血液供應(yīng)豐富,腫瘤生長較大,可以有利于觀察磁流體熱療治療對腫瘤體積變化的影響。納米粒子的磁流體熱療的應(yīng)用已被證明在幾種動(dòng)物模型中能夠抑制腫瘤的生長,然而,使用磁流體熱療在體內(nèi)治療舌癌的可行性是不確切的,其機(jī)制也尚不清楚。在目前研究中,本課題旨在建立裸鼠腋窩皮下舌癌動(dòng)物模型,瘤內(nèi)注射納米磁流體后,在交變磁場下熱療治療,觀察磁流體熱療對腫瘤生長的影響,進(jìn)一步驗(yàn)證納米磁流體熱療治療惡性腫瘤的可行性及臨床應(yīng)用價(jià)值。 目的： 檢測二氧化硅包裹的Mn0.4Zn0.6Fe2O4納米磁流體在交變磁場下的體內(nèi)外升溫性能,建立Tca8113人舌鱗癌細(xì)胞裸鼠皮下移植瘤模型,探討Mn0.4Zn0.6Fe2O4納米磁流體局部熱療治療裸鼠Tca8113舌癌模型的可行性及其對瘤體生長的影響。 方法： 1.Tca8113細(xì)胞的培養(yǎng)：復(fù)蘇舌癌細(xì)胞株Tca8113細(xì)胞,RPMI160培養(yǎng)基體外培養(yǎng),觀察Tca8113細(xì)胞的形態(tài)及增值活性。 2.將Mn0.4Zn0.6Fe2O4納米磁流體進(jìn)行透射電子顯微鏡檢測、磁滯回線檢測以及體外不同磁場電流強(qiáng)度下的升溫實(shí)驗(yàn)檢測。 3.人舌癌Tca8113細(xì)胞對Mn0.4Zn0.6Fe2O4納米磁顆粒的吞噬實(shí)驗(yàn)：取處于對數(shù)生長期的Tca8113細(xì)胞,與加入含有二氧化硅包裹的錳鋅鐵氧體納米顆粒的培養(yǎng)基共培養(yǎng)24h,普魯士藍(lán)染色,倒置顯微鏡下觀察細(xì)胞對納米顆粒的吞噬情況。 4.Tca8113細(xì)胞毒性檢測及細(xì)胞形態(tài)學(xué)觀察：將含有不同濃度納米粒子浸提液的RPMI-1640培養(yǎng)基與Tca8113細(xì)胞共同培養(yǎng),觀察細(xì)胞形態(tài)學(xué)的改變,檢測細(xì)胞增殖活性的改變,以及對細(xì)胞的毒性檢測。 5.體內(nèi)試驗(yàn)：隨機(jī)選擇BALB/c-nu雄性裸鼠27只,人舌鱗癌細(xì)胞Tca8113腋窩皮下注射建立裸鼠腫瘤模型。將接種10天的荷瘤鼠隨機(jī)分為5組：即空白對照組(A組)、磁流體對照組(B組)、磁場對照組(C組)、磁流體熱療1組(D組)、磁流體熱療2組(E組)。磁流體熱療1組和磁流體熱療2組直接瘤內(nèi)注射磁流體24h后暴露于交變磁場,用熱敏電偶溫度傳感器測量交變磁場下瘤體中心、瘤體邊緣及肛周的溫度變化。當(dāng)實(shí)驗(yàn)組腫瘤中心區(qū)溫度穩(wěn)定在43℃后,再持續(xù)熱療30min。磁流體熱療2組在5～7后再重復(fù)加熱一次。磁場對照組加熱時(shí)間及加熱條件與磁流體熱療組完全相同。而空白對照組不經(jīng)任何處理,磁流體對照組只瘤內(nèi)注射納米磁流體未經(jīng)磁場加熱。熱療治療結(jié)束后24h,各組隨機(jī)選擇1只裸鼠,麻醉注射過量處死,迅速取出腫瘤組織、周圍肌肉組織、心臟、肺、肝臟、脾臟和腎臟,肉眼大體觀察組織的病理改變。剖開即刻用4%甲醛固定,石蠟包埋切片,HE和普魯士藍(lán)染色,光學(xué)顯微鏡下觀察腫瘤組織病理學(xué)變化及重要臟器內(nèi)有無納米磁流體殘留。九周后處死所有實(shí)驗(yàn)組裸鼠,測量瘤體體積,計(jì)算腫瘤體積抑制率,肉眼大體觀察標(biāo)本并進(jìn)行組織病理學(xué)檢查。 6.統(tǒng)計(jì)學(xué)分析：統(tǒng)計(jì)學(xué)分析采用SPSS20.0軟件包處理。計(jì)量數(shù)據(jù)采用均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,吸光度值、腫瘤體積、腫瘤最長徑和各組溫度采用完全隨機(jī)設(shè)計(jì)資料的單因素方差分析,進(jìn)一步采用L.S.D.法做組間、組內(nèi)兩兩之間比較。P0.05為差異具有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1.Tca8113細(xì)胞的培養(yǎng)：Tca8113細(xì)胞形態(tài)符合人舌鱗癌細(xì)胞形態(tài)特征,在倒置顯微鏡下觀察可發(fā)現(xiàn),單個(gè)細(xì)胞呈長梭形或多角形,呈集落性生長,增殖較快,約第二天開始進(jìn)入對數(shù)生長期,第四天進(jìn)入平臺期。 2.在透射電子顯微鏡下觀察拍照發(fā)現(xiàn)納米粒子顆粒在水溶液中分散較好,粒徑約10nm,沒有聚集現(xiàn)象發(fā)生；通過磁滯回線檢測,錳鋅鐵氧體納米粒子具有超順磁性。體外磁流體升溫實(shí)驗(yàn)檢測發(fā)現(xiàn)納米磁流體升溫速率隨磁流體濃度增大而增大,在電流為50A和磁場頻率為237KHZ時(shí),磁流體可在20min內(nèi)快速上升至80℃變緩慢最后維持在83℃不上升。 3.Tca8113細(xì)胞與Mn0.4Zn0.6Fe2O4顆粒共培養(yǎng)24h后,倒置顯微鏡觀察發(fā)現(xiàn),普魯士藍(lán)染色法,Tca8113細(xì)胞內(nèi)有呈藍(lán)色改變的氧化鐵顆粒,而空白對照組無藍(lán)色顆粒。 4.25%、50%、75%浸提液組分別與Tca8113細(xì)胞共培養(yǎng),細(xì)胞數(shù)量隨著培養(yǎng)時(shí)間的延長細(xì)胞數(shù)量逐漸增多,細(xì)胞形態(tài)未見明顯變化,無細(xì)胞毒性；100%浸提液組生長緩慢,可見少量細(xì)胞核固縮,表現(xiàn)為輕微的細(xì)胞毒性。隨著浸提液濃度的增高,MTT檢測細(xì)胞的增殖活性明顯降低。 5.裸鼠接種10d時(shí)腋窩皮下瘤體直徑約0.8cm-lcm,裸鼠成瘤率100%。隨機(jī)分為5組,熱療前各組裸鼠瘤體體積之間比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。磁流體熱療組腫瘤中心溫度可相對穩(wěn)定在43℃,瘤體邊緣區(qū)溫度比瘤體中心區(qū)溫度低2-3℃約40℃,直腸幾乎不升溫。磁場對照組無升溫現(xiàn)象,腫瘤中心溫度與體溫相同。空白對照組和磁流體對照組裸鼠腫瘤中心溫度穩(wěn)定在30.8℃,也無升溫現(xiàn)象。磁流體熱療1組和磁流體熱療2組腫瘤中心區(qū)、腫瘤邊緣區(qū)溫度與空白對照組進(jìn)行比較,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。磁流體熱療1組與磁流體熱療2組之間進(jìn)行比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。磁流體熱療組腫瘤中心區(qū)與腫瘤邊緣區(qū)之間比較,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。所有實(shí)驗(yàn)裸鼠均能耐受熱療。各組裸鼠經(jīng)過熱療治療后瘤體體積均呈一定程度的增大趨勢�？瞻讓φ战M和磁場對照組增大趨勢較為明顯,磁流體對照組增長趨勢稍低。磁場對照組、磁流體對照組、空白對照組即三對照組間腫瘤體積進(jìn)行比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)；磁流體熱療組裸鼠處理后瘤體體積6周內(nèi)即種瘤后第2、3、4、5周腫瘤體積與對照組之間進(jìn)行比較,差異具有統(tǒng)計(jì)學(xué)意義(P0.05),尤其是磁流體熱療2組腫瘤體積與各對照組之間比較差異更為顯著(P0.01)；兩磁流體熱療組在種瘤后第3、4、5周腫瘤體積比較差異亦具有統(tǒng)計(jì)學(xué)意義(P0.05),但6周之后由于熱療瘤體周邊殘留的腫瘤組織細(xì)胞繼續(xù)增長,6周后各組腫瘤體積之間進(jìn)行比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。磁流體熱療1組和磁流體熱療2組瘤內(nèi)注射磁流體熱療治療后,腫瘤生長受到抑制,磁流體熱療2組抑制作用更為明顯,磁流體熱療1組與磁流體熱療2組在種瘤后2-5周腫瘤體積抑制率分別為23.80%～41.81%和34.92%～56.76%。各組心臟、肺、肝臟、腎臟和脾臟等重要器官未見有腫瘤細(xì)胞的轉(zhuǎn)移,也未見納米磁流體的殘留,無明顯異常改變。 結(jié)論： 1.Mn0.4Zn0.6Fe2O4納米磁流體水溶性好,具有磁性；Mn0.4Zn0.6Fe2O4納米磁流體在體內(nèi)外均具有良好升溫性能的特性,在體內(nèi)可使腫瘤區(qū)域獲得比較均勻分布的43℃熱療治療溫度,而其余正常組織不升溫。 2.人舌Tca8113細(xì)胞可吞噬Mn0.4Zn0.6Fe2O4納米顆粒。 3.Mn0.4Zn0.6Fe2O4磁流體對細(xì)胞無明顯細(xì)胞毒性,毒性與磁流體濃度密切相關(guān)；且磁流體對舌癌Tca8113細(xì)胞形態(tài)沒有明顯的影響。 4.成功構(gòu)建舌癌動(dòng)物模型,組織病理學(xué)觀察確定為Tca8113鱗狀細(xì)胞癌。43℃磁流體熱療可導(dǎo)致腫瘤組織明顯壞死,顯著抑制腫瘤生長；同樣,磁流體熱療可促進(jìn)瘤內(nèi)納米磁流體的分散,實(shí)現(xiàn)熱療的周邊效應(yīng)。磁流體熱療對腫瘤組織細(xì)胞的殺傷作用與熱療頻率有關(guān)。一次性瘤內(nèi)注射納米磁流體后,磁流體介導(dǎo)的43℃熱療治療裸鼠Tca8113皮下移植瘤安全、有效、可行、靶向性好,低副作用,且可進(jìn)行重復(fù)熱療,治療腫瘤具有潛在的臨床應(yīng)有價(jià)值。
[Abstract]:Oral squamous cell carcinoma (OSSC) is the most common malignant tumor in the oral and maxillofacial region. It comes from the complex squamous cells of the skin and mucosa. In oral squamous cell carcinoma, one of the most common types of oral squamous cell carcinoma is the squamous cell carcinoma of the tongue. Age is also becoming younger. Finding active and effective ways of treatment has become an urgent and important task. Surgery, chemotherapy, and radiotherapy are still the three most important methods for the treatment of squamous cell carcinoma of the tongue, but the damage and risk of surgical treatment and the huge toxic and side effects of radiotherapy and chemotherapy have seriously affected the cancer patients. Therefore, it is imperative to study and develop a new approach to the treatment of squamous cell carcinoma of the tongue, which is targeted, safer, more effective, and less side effects, without invasive or minimally invasive tongue squamous cell carcinoma.
Using the principle that tumor tissue is more sensitive to high temperature than normal tissue, the tumor tissue is heated to more than 43 degrees centigrade, and the tumor cells are not tolerable to the normal cells, which can not tolerate the continuous high temperature, thus the different degrees of apoptosis or necrosis occur. The method of treating malignant tumor is called Hyperthermia. In recent years, the rapid development of nanotechnology has made it make it For a challenging innovation in the medical field, Magnetic fluid hyperthermia (MFH) is a magnetic fluid that has modified or added some kind of antibody to the inside of the tumor via an intravenous or direct injection. The magnetic fluid is phagocytic or deposited in the cell between the tumor cells, in the Alternating magnetic field (Alternating magnetic field, AMF) the magnetic nanoparticles rotate through the magnetic vector and the physical rotation of the particles themselves, that is, the tumor cells are killed by Nair relaxation and the Brown relaxation mechanism, and the normal tissue around the normal tissue is not obviously heated and highly targeted. The emergence of magnetic fluid thermotherapy technology solves the problem of the accurate location of the focus in the hyperthermia, and it is swollen in the swelling. The magnetic fluid thermotherapy is a new method for the hyperthermia of the deep tissue. It can be obtained by regulating the concentration of magnetic fluid or the intensity of the magnetic field. This experiment is achieved by adjusting the concentration of magnetic fluid; the local thermotherapy can be divided into thermotherapy (42-46), high Hyperthermia (46-70 C), hot excision (above 70 degrees C). Compared with the traditional thermotherapy, such as infrared thermotherapy, ultrasound thermotherapy, microwave hyperthermia and radiofrequency ablation, electromagnetic wave thermotherapy, MFH technology has the characteristics of treatment of deep tumor, small side effect, minimally invasive and even noninvasive, and the tumor site is injected with nanoscale magnetic fluid, which can be used for patients. Repeated hyperthermia were repeated.
The immune deficiency of nude mice, under certain conditions, does not exclude tissue transplantation from different species of animals; the transplanted tumor remains the original histologic structure and biological characteristics; at the same time, the metastasis of the transplanted tumor is almost rare. These conditions are the basis of the successful establishment of the tumor model of the cancer of the tongue, and also an important guarantee for this experiment. The tumor tissue growth table is easy to be palpable in vitro, rich in blood supply and large growth of tumor, which can help observe the effect of magnetic fluid thermotherapy on tumor volume change. The application of magnetic fluid thermotherapy for nanoparticles has been proved to be able to inhibit tumor growth in several animal models, however, however, The feasibility of using magnetic fluid thermotherapy in the treatment of tongue cancer in vivo is not accurate and its mechanism is still unclear. In the present study, the aim of this study is to establish an animal model of subcutaneous tongue cancer in nude mice. After intratumoral injection of magnetic fluid, the effect of thermotherapy on the growth of tumor is observed under the alternating magnetic field and the effect of magnetic fluid thermotherapy on tumor growth is observed. The feasibility and clinical application value of M magnetic fluid hyperthermia in the treatment of malignant tumors.
Objective:
To detect the heating performance of Mn0.4Zn0.6Fe2O4 nano magnetic fluid encapsulated by silica in the alternating magnetic field in vivo and in vitro, the model of subcutaneous transplantation of Tca8113 human tongue squamous cell carcinoma cells in nude mice was established, and the feasibility of Mn0.4Zn0.6Fe2O4 nano magnetic fluid local thermotherapy in the treatment of Tca8113 tongue cancer model in nude mice and its effect on the growth of the tumor were discussed.
Method錛，

本文編號：1914833