本文選題：喉癌 + 表皮生長(zhǎng)因子受體��； 參考：《昆明醫(yī)科大學(xué)》2012年博士論文

【摘要】：第一部分表皮生長(zhǎng)因子受體在喉鱗癌組織中的表達(dá)及siRNA沉默EGFR基因誘導(dǎo)Hep-2細(xì)胞凋亡研究 目的：檢測(cè)喉鱗癌組織中表皮生長(zhǎng)因子受體(EGFR)的表達(dá),探討其在喉癌發(fā)生中的作用。運(yùn)用RNA干擾技術(shù)研究EGFR表達(dá)下調(diào)誘導(dǎo)Hep-2細(xì)胞凋亡情況。 方法：收集2008年1月～2011年3月昆明醫(yī)學(xué)院第一附屬醫(yī)院、第三附屬醫(yī)院頭頸外科36例喉鱗癌組織標(biāo)本,正常對(duì)照來(lái)自其中的13例喉全切除術(shù)標(biāo)本,陽(yáng)性對(duì)照選取Hep-2細(xì)胞株。運(yùn)用Western Blot技術(shù)檢測(cè)喉癌組織、癌旁組織及正常粘膜組織EGFR的表達(dá),圖像結(jié)果進(jìn)行Quantity One軟件定量分析,得出EGFR蛋白相對(duì)表達(dá)量,分析其與癌組織病理分級(jí)的關(guān)系。進(jìn)而運(yùn)用siRNA技術(shù)沉默Hep-2細(xì)胞EGFR基因,RT-PCR及Western Blot檢測(cè)EGFR基因表達(dá)抑制效果,流式細(xì)胞術(shù)進(jìn)一步檢測(cè)EGFRsiRNA質(zhì)粒轉(zhuǎn)染48h時(shí)Hep-2細(xì)胞凋亡情況。 結(jié)果：(1)喉癌標(biāo)本中EGFR蛋白的表達(dá)從高到低依次為喉鱗癌組織、癌旁組織,正常粘膜組織幾乎無(wú)條帶顯示。喉癌組織、癌旁組織不同病理分級(jí)之間EGFR相對(duì)表達(dá)量差異有統(tǒng)計(jì)學(xué)意義,P0.05。相同病理分級(jí)喉癌組織、癌旁組織及正常組織之間EGFR相對(duì)表達(dá)量差異有高度統(tǒng)計(jì)學(xué)意義,P0.01。(2)成功獲得EGFRsiRNA表達(dá)質(zhì)粒pSi-hEGFR,轉(zhuǎn)染Hep-2細(xì)胞48h后RT-PCR檢測(cè)結(jié)果顯示,EGFRmRNA表達(dá)水平下調(diào)46%,Western Blot檢測(cè)獲得一致的結(jié)果。pSi-hEGFR質(zhì)粒轉(zhuǎn)染Hep-2細(xì)胞48h后流式細(xì)胞術(shù)檢測(cè)發(fā)現(xiàn)：EGFR陽(yáng)性表達(dá)抑制率為83.8%；細(xì)胞凋亡率達(dá)(15.13±2.15)%,而pSi-Negative質(zhì)粒轉(zhuǎn)染組凋亡率為(7.03±1.08)%,差異有高度統(tǒng)計(jì)學(xué)意義,P0.01。 結(jié)論：EGFR在喉癌組織中存在過(guò)表達(dá)現(xiàn)象,與喉癌組織不同部位及病理分級(jí)有相關(guān)性。siRNA沉默EGFR基因可以下調(diào)Hep-2細(xì)胞EGFR mRNA和蛋白水平表達(dá),同時(shí)發(fā)現(xiàn)可以誘導(dǎo)Hep-2細(xì)胞出現(xiàn)凋亡。本研究提示EGFR可能參與喉癌的發(fā)生,其表達(dá)下調(diào)可以起到部分抑瘤作用,為以EGFR為靶點(diǎn)的抗腫瘤治療方法的探索提供基礎(chǔ)理論依據(jù)。 第二部分EGFRmAb功能化修飾金納米棒光熱作用抑制Hep-2細(xì)胞增殖研究 目的：用EGFRmAb功能化修飾金納米棒(AuNRs)獲得穩(wěn)定的EGFRmAb/AuNRs共軛物,探索AuNRs進(jìn)入Hep-2細(xì)胞的機(jī)制,了解AuNRs光熱轉(zhuǎn)換的升溫規(guī)律,評(píng)價(jià)AuNRs的生物安全性,觀察EGFRmAb功能化修飾AuNRs靶向光熱效應(yīng)抑制Hep-2細(xì)胞增殖作用。 方法：合成EGFRmAb/AuNRs后用透射電鏡進(jìn)行表征,用紫外-可見(jiàn)-近紅外光譜儀對(duì)其進(jìn)行光譜分析。將AuNRs、EGFRmAb/AuNRs分別與Hep-2細(xì)胞共孵育,透射電鏡觀察AuNRs進(jìn)入細(xì)胞及細(xì)胞內(nèi)分布。采用ICP-AES元素分析法檢測(cè)EGFRmAb介導(dǎo)下AuNRs進(jìn)入Hep-2細(xì)胞的量。NIR激光照射后用溫度計(jì)測(cè)量不同狀況下AuNRs溶液溫度上升規(guī)律。用MTT法檢測(cè)不同濃度AuNRs細(xì)胞毒性。用臺(tái)盼藍(lán)染色法大體觀察AuNRs光熱作用殺傷腫瘤細(xì)胞現(xiàn)象,繼而用MTT法檢測(cè)不同功率NIR激光照射AuNRs后不同時(shí)間點(diǎn)Hep-2細(xì)胞的增殖抑制率,比較AuNRs和EGFRmAb/AuNRs光熱作用的抑瘤效果。 結(jié)果：(1)電鏡表征顯示本實(shí)驗(yàn)用AuNRs具有良好的穩(wěn)定性和分散性,微粒大小均勻,長(zhǎng)徑均值為49.81nm,橫徑均值為12.70nm,長(zhǎng)橫比均值為3.92。紫外-可見(jiàn)-近紅外光譜儀檢測(cè)顯示,AuNRs溶液表現(xiàn)出雙共振吸收峰,橫向?yàn)?10nm,縱向?yàn)?00nm。AuNRs經(jīng)EGFRmAb修飾后縱向共振吸收峰位紅移7-9nm,并不明顯改變?cè)泄鈱W(xué)特性。(2)電鏡觀察顯示：AuNRs和Hep-2細(xì)胞共同孵育30min時(shí),可見(jiàn)AuNRs于細(xì)胞膜表面,出現(xiàn)胞吞現(xiàn)象；3h時(shí)可見(jiàn)AuNRs成簇狀位于溶酶體內(nèi)；6h時(shí)可見(jiàn)AuNRs同時(shí)存在于細(xì)胞質(zhì)、溶酶體和線粒體內(nèi),少量AuNRs貼近核膜；未發(fā)現(xiàn)細(xì)胞形態(tài)和細(xì)胞器(線粒體)結(jié)構(gòu)明顯改變。EGFRmAb功能化修飾的AuNRs進(jìn)入細(xì)胞的數(shù)量比未修飾的要多,進(jìn)一步ICP-AES元素分析顯示共孵育6h時(shí)EGFRmAb功能化修飾明顯提高AuNRs進(jìn)入細(xì)胞的數(shù)量,增加48.14%。(3)NIR激光照射AuNRs溶液可以引起溫度迅速升高,溫度升高的幅度和AuNRs濃度、照射功率及容器容積有一定關(guān)系。研究發(fā)現(xiàn),在室溫為23℃情況下,用3.0W/cm2功率NIR激光照射0.1nmol/L AuNRs溶液(1ml/每孔,24孔板)4min時(shí)溫度達(dá)到46℃,隨后穩(wěn)定,滿足本實(shí)驗(yàn)光熱誘導(dǎo)凋亡的溫度要求。(4)MTT法檢測(cè)顯示AuNRs在0.1nmol/L～2.0nmol/L濃度范圍內(nèi)未出現(xiàn)細(xì)胞活力明顯下降,AuNRs/EGFRmAb共軛物對(duì)Hep-2細(xì)胞活力沒(méi)有明顯影響。(5)5.2W/cm2功率NIR激光照射AuNRs和Hep-2細(xì)胞共孵育培養(yǎng)液8min后臺(tái)盼藍(lán)染色顯示細(xì)胞全部死亡。進(jìn)一步MTT法檢測(cè)顯示：AuNRs和AuNRs/EGFRmAb光熱作用可以明顯引起Hep-2細(xì)胞增殖抑制,呈激光功率劑量依賴(lài)性；而單純NIR激光照射及EGFR單抗對(duì)Hep-2細(xì)胞沒(méi)有明顯的增殖抑制作用；控制NIR激光功率為3.0W/cm2,則Hep-2細(xì)胞的增殖抑制呈明顯的時(shí)間效應(yīng)關(guān)系,72h抑制率最高,達(dá)69.24%；同時(shí)發(fā)現(xiàn),AuNRs/EGFRmAb光熱效應(yīng)對(duì)Hep-2細(xì)胞的增殖抑制作用明顯高于未修飾AuNRs, P0.01. 結(jié)論：EGFRmAb修飾AuNRs后未明顯改變?cè)械男螤詈凸鈱W(xué)性質(zhì),但可以明顯增加AuNRs進(jìn)入Hep-2細(xì)胞的能力,推測(cè)可能和EGFR介導(dǎo)的胞吞有關(guān)。AuNRs進(jìn)入細(xì)胞后主要存在于細(xì)胞質(zhì)、溶酶體和線粒體中,其本身并沒(méi)用明顯的細(xì)胞毒性,但在NIR激光的照射下,能迅速引起環(huán)境溫度的升高,導(dǎo)致細(xì)胞出現(xiàn)增殖抑制或死亡�？刂芅IR激光的照射功率,細(xì)胞的增殖抑制呈時(shí)效性。EGFRmAb功能化修飾明顯提高AuNRs光熱抑瘤效果,而單純的NIR激光照射和EGFRmAb并沒(méi)有明顯的細(xì)胞增殖抑制現(xiàn)象。由于AuNRs位于細(xì)胞內(nèi)線粒體中,我們推測(cè)上述細(xì)胞增殖抑制可能是AuNRs光熱誘導(dǎo)的內(nèi)源性凋亡導(dǎo)致的。 第三部分EGFRmAb/AuNRs光熱誘導(dǎo)Hep-2細(xì)胞凋亡機(jī)制研究 目的：控制NIR激光照射參數(shù),觀察EGFRmAb/AuNRs光熱誘導(dǎo)Hep-2細(xì)胞凋亡現(xiàn)象,通過(guò)檢測(cè)線粒體途徑凋亡相關(guān)蛋白分子表達(dá)來(lái)闡述凋亡分子機(jī)制,同時(shí)證明EGFRmAb功能化修飾能夠有效提高AuNRs光熱誘導(dǎo)Hep-2細(xì)胞凋亡效果。 方法：參照第二部分實(shí)驗(yàn),控制NIR激光照射參數(shù)為：功率3.0W/cm2,時(shí)間8min。電鏡觀察照射后Hep-2細(xì)胞超微結(jié)構(gòu)變化及凋亡。TUNEL/PI雙染法流式細(xì)胞術(shù)檢測(cè)照射后不同時(shí)間點(diǎn)細(xì)胞凋亡和周期,進(jìn)而流式細(xì)胞術(shù)檢測(cè)照射后不同時(shí)間點(diǎn)Hep-2細(xì)胞活化Caspase-3、Bcl-2/Bax和Cyt-c陽(yáng)性表達(dá)率、細(xì)胞內(nèi)ROS水平和Ca2+濃度及細(xì)胞線粒體膜電位(△甲m)下降率。 結(jié)果：(1)照射后48h電鏡觀察NIR+AuNRs組和NIR+EGFRmAb/AuNRs組可見(jiàn)細(xì)胞凋亡。(2)NIR+AuNRs組和NIR+EGFRmAb/AuNRs組細(xì)胞在照射后24h、48h時(shí)表現(xiàn)出明顯的G0/G1期阻滯,同時(shí)S期比率下降,并呈時(shí)間效應(yīng)關(guān)系。在照射后48h時(shí),EGFRmAb/AuNRs組和AuNRs組相比,S期比率下降相對(duì)明顯,P0.01。(3)NIR+AuNRs組與NIR+EGFRmAb/AuNRs組細(xì)胞在照射后出現(xiàn)明顯凋亡,呈時(shí)間效應(yīng)關(guān)系。NIR+EGFRmAb/AuNRs組細(xì)胞在照射后三個(gè)時(shí)間點(diǎn)(24h、48h、72h)的凋亡：率均比NIR+AuNRs組要高,P0.05。照射后72h時(shí)NIR+EGFRmAb/AuNRs組細(xì)胞凋亡率最高,達(dá)73.63%。(4)照射后NIR+AuNRs組和NIR+EGFRmAb/AuNRs組細(xì)胞活化Caspase-3、Bax陽(yáng)性表達(dá)率、△Ψm下降率及細(xì)胞內(nèi)ROS水平、Ca2+濃度升高,細(xì)胞Bcl-2陽(yáng)性表達(dá)率降低。隨時(shí)間推移上述指標(biāo)各自變化趨勢(shì)明顯,但NIR+EGFRmAb/AuNRs組細(xì)胞上述指標(biāo)改變比NIR+AuNRs組更為明顯,P0.05。照射后NIR+AuNRs組與NIR+EGFRmAb/AuNRs組細(xì)胞Cyt-c陽(yáng)性表達(dá)率明顯升高。照射后1h時(shí)檢出Cyt-c陽(yáng)性表達(dá),24h時(shí)最高,隨后呈逐漸下降趨勢(shì)。在照射后24h、48h時(shí),NIR+EGFRmAb/AuNRs組細(xì)胞Cyt-c陽(yáng)性表達(dá)率和NIR+AuNRs組比較升高更為明顯,P0.01。 結(jié)論：控制合適的NIR激光照射參數(shù)可以通過(guò)AuNRs光熱作用誘導(dǎo)Hep-2細(xì)胞出現(xiàn)凋亡,這種凋亡和細(xì)胞周期阻滯密切相關(guān)。ROS和Ca2+含量升高、△ψm下降、Cyt-c釋放、活化Caspase-3上調(diào)及Bcl-2/Bax比例下降在上述凋亡過(guò)程中起重要作用,可以看出,線粒體介導(dǎo)的內(nèi)源性途徑是AuNRs光熱誘導(dǎo)Hep-2細(xì)胞凋亡的重要方式。EGFRmAb功能化修飾可以通過(guò)AuNRs的靶向聚集而提高誘導(dǎo)凋亡的效果,但EGFR單抗本身并沒(méi)有明顯參與凋亡的發(fā)生。 第四部分EGFRmAb/AuNRs光熱治療裸鼠喉鱗癌移植瘤研究 目的：觀察局部注射及全身靜脈給藥模式下AuNRs光熱治療裸鼠皮下喉癌移植瘤效果并檢測(cè)治療后腫瘤細(xì)胞凋亡情況。 方法：建立裸鼠皮下喉癌移植瘤模型。瘤體局部注射AuNRs,經(jīng)尾靜脈全身給EGFRmAb/AuNRs,NIR激光照射參數(shù)同第三部分體外實(shí)驗(yàn),照射過(guò)程中測(cè)量腫瘤局部皮溫變化,游標(biāo)卡尺測(cè)量照射后腫瘤大小變化。照射后2周結(jié)束實(shí)驗(yàn),繪制移植瘤生長(zhǎng)曲線,裸鼠處死后切取腫瘤標(biāo)本,TUNEL/PI雙染法流式細(xì)胞術(shù)檢測(cè)腫瘤細(xì)胞凋亡和壞死。 結(jié)果：NIR激光照射3min后腫瘤處皮膚溫度趨于穩(wěn)定,AuNRs局部注射組照射后溫度上升15℃,EGFRmAb/AuNRs靜脈給藥組照射后溫度上升11℃。兩觀察組照射后腫瘤的生長(zhǎng)均有明顯的抑制現(xiàn)象,各觀察時(shí)間點(diǎn)生長(zhǎng)速度明顯變緩,但AuNRs局部注射組照射后腫瘤抑制效果更明顯一些,P0.05。單獨(dú)EGFRmAb(E3138,Sigma公司)靜脈給藥、小劑量NIR激光照射及不經(jīng)EGFRmAb修飾的AuNRs靜脈給藥后照射均沒(méi)有產(chǎn)生明顯的腫瘤抑制效果。流式細(xì)胞術(shù)檢測(cè)發(fā)現(xiàn),AuNRs局部注射組及EGFRmAb/AuNRs靜脈給藥組照射后2周腫瘤組織細(xì)胞出現(xiàn)明顯的凋亡,凋亡率分別為58.35%和30.46%。AuNRs局部注射組照射后凋亡率和壞死率均明顯高于EGFRmAb/AuNRs靜脈給藥組,P0.01。 結(jié)論：AuNRs直接瘤體注射光熱作用可以明顯抑制裸鼠喉癌移植瘤的生長(zhǎng),EGFRmAb/AuNRs靜脈給藥也可以起到一定的治療效果,流式細(xì)胞術(shù)檢測(cè)印證了光熱誘導(dǎo)凋亡現(xiàn)象的存在。動(dòng)物實(shí)驗(yàn)的效果給EGFRmAb/AuNRs體內(nèi)靶向光熱治療腫瘤帶來(lái)希望,由于體內(nèi)環(huán)境的復(fù)雜性,要真正實(shí)現(xiàn)體內(nèi)深層腫瘤安全有效的AuNRs光熱治療還需進(jìn)一步深入研究。
[Abstract]:Part one expression of epidermal growth factor receptor in laryngeal squamous cell carcinoma and apoptosis of Hep-2 cells induced by siRNA silencing EGFR gene
Objective: to detect the expression of epidermal growth factor receptor (EGFR) in the tissues of laryngeal squamous cell carcinoma (LSCC) and explore its role in the development of larynx cancer. RNA interference technique was used to study the down regulation of EGFR expression to induce apoptosis in Hep-2 cells.
Methods: from January 2008 to March 2011, 36 cases of laryngeal squamous cell carcinoma were collected from the First Affiliated Hospital of Kunming Medical University, third affiliated hospitals, and 13 cases of laryngeal squamous cell carcinoma were collected from normal controls. The positive controls were selected from 13 cases of total laryngectomy. Western Blot technique was used to detect the tissues of the larynx, the para cancerous tissue and the normal mucous tissue EGFR By quantitative analysis of Quantity One software, the relative expression of EGFR protein was obtained, and the relationship between EGFR protein and histopathological classification was analyzed. Then siRNA technique was used to silence Hep-2 cell EGFR gene, RT-PCR and Western Blot were used to detect the inhibitory effect of EGFR gene expression. Flow cytometry further detected EGFRsiRNA plasmid transfection 48h. Apoptosis of EP-2 cells.
Results: (1) the expression of EGFR protein in the laryngeal carcinoma specimens from high to low was in the order of laryngeal squamous cell carcinoma, the para cancerous tissue, and the normal mucosal tissue almost no strip display. The relative expression of EGFR in the larynx tissues and the para cancerous tissues was statistically significant. P0.05. was the same pathological classification of the larynx, para cancer tissue and normal tissue. The relative expression of EGFR was statistically significant. P0.01. (2) successfully obtained the EGFRsiRNA expression plasmid pSi-hEGFR. The RT-PCR detection results of 48h transfected Hep-2 cells showed that the expression level of EGFRmRNA was down down by 46%, and the Western Blot detection obtained the same result of the.PSi-hEGFR plasmid transfected to the Hep-2 cells after the flow cytometry. The inhibition rate of expression was 83.8%, the apoptosis rate was (15.13 + 2.15)%, while the apoptosis rate of pSi-Negative plasmid transfected group was (7.03 + 1.08)%, and the difference was statistically significant, P0.01.
Conclusion: there is a phenomenon of overexpression of EGFR in the carcinoma of the larynx..siRNA silencing EGFR gene can down regulate the expression of EGFR mRNA and protein in Hep-2 cells and induce apoptosis of Hep-2 cells. This study suggests that EGFR may be involved in the occurrence of laryngeal carcinoma and its expression can be down regulated. It plays a part of tumor suppressive effect, and provides a basic theoretical basis for the exploration of anti-tumor therapy targeting EGFR.
The second part of EGFRmAb functionalized gold nanorods inhibit the proliferation of Hep-2 cells by photothermal action.
Objective: to obtain stable EGFRmAb/AuNRs conjugates with EGFRmAb functionalized gold nanorods (AuNRs), explore the mechanism of AuNRs into Hep-2 cells, understand the heating law of AuNRs photothermal transformation, evaluate the biosafety of AuNRs, and observe the function of EGFRmAb functionalized modified AuNRs target to inhibit the proliferation of Hep-2 cells.
Methods: the EGFRmAb/AuNRs was characterized by transmission electron microscopy, and the spectral analysis was carried out by ultraviolet visible near infrared spectrometer. AuNRs, EGFRmAb/AuNRs were incubated with Hep-2 cells respectively. The distribution of AuNRs into cells and cells was observed by transmission electron microscopy. ICP-AES element analysis was used to detect AuNRs into Hep-2 cells under EGFRmAb. The temperature rise of AuNRs solution under different conditions was measured by a thermometer after.NIR laser irradiation. The cytotoxicity of AuNRs cells at different concentrations was detected by MTT method. The phenomenon of AuNRs photothermal effect on tumor cells was observed by trypan blue staining, and then MTT method was used to detect the proliferation of Hep-2 cells at different time points of different power NIR lasers at different time points. The inhibition rate was compared with the antitumor effect of AuNRs and EGFRmAb/AuNRs photothermal therapy.
Results: (1) the electron microscopic characterization showed that the experiment with AuNRs had good stability and dispersion, the particle size was uniform, the mean length of diameter was 49.81nm, the mean diameter of the transverse diameter was 12.70nm, the mean value of the long transverse ratio was 3.92. ultraviolet visible near infrared spectrometer, and the AuNRs solution showed the double resonance absorption peak, the transverse 510nm, and the longitudinal 800nm.AuNRs via EGFRm. The longitudinal resonance absorption peak was red shift 7-9nm after Ab modification. (2) electron microscope observation showed that when AuNRs and Hep-2 cells incubated 30min together, AuNRs was found on the surface of the cell membrane, and there was a phenomenon of endocytosis; AuNRs was clustered in the soluble enzyme in 3h, and AuNRs was found at the same time at the cytoplasm, lysosome and. In mitochondria, a small amount of AuNRs was close to the nuclear membrane; no cell morphology and organelles (mitochondria) structure obviously changed the number of.EGFRmAb functionalized AuNRs to enter cells more than that of unmodified ones. Further ICP-AES analysis showed that EGFRmAb functional modification significantly increased the number of AuNRs into cells and increased 48.14%. (3). NIR laser irradiation of AuNRs solution can cause a rapid increase in temperature. The amplitude of the temperature rise and the concentration of AuNRs, the power of the irradiation and the volume of the container have a certain relation. The study found that the temperature of 0.1nmol/L AuNRs solution (1ml/ per pore, 24 hole plate) at 4min at room temperature is 23 C at room temperature, and the temperature reaches 46 degrees C, then stable, and satisfies this. Temperature requirements for apoptosis induced by light and heat. (4) MTT assay showed that AuNRs did not markedly decrease cell viability in 0.1nmol/L ~ 2.0nmol/L concentration, and AuNRs/EGFRmAb conjugates had no significant influence on Hep-2 cell viability. (5) 5.2W/cm2 power NIR laser irradiation of AuNRs and Hep-2 cell co incubating medium was shown by 8min background blue staining All the cells died. Further MTT assay showed that the photothermal effect of AuNRs and AuNRs/EGFRmAb could obviously induce the proliferation inhibition of Hep-2 cells, and the laser power dose depended, while the pure NIR laser irradiation and the EGFR mAb did not inhibit the proliferation of Hep-2 cells, and the NIR laser power was 3.0W/cm2, then the Hep-2 cells increased. The inhibitory rate of 72h was the highest, up to 69.24%, and the inhibitory effect of AuNRs/EGFRmAb photothermal effect on the proliferation of Hep-2 cells was significantly higher than that of unmodified AuNRs, P0.01.
Conclusion: EGFRmAb modified AuNRs did not obviously change the original shape and optical properties, but it could significantly increase the ability of AuNRs to enter Hep-2 cells. It is presumed that EGFR mediated cytosol related.AuNRs enters the cell and is mainly present in the cytoplasm, lysosomes and mitochondria, and its body does not use obvious cytotoxicity, but in NIR laser Under the irradiation, it can cause the increase of the temperature of the environment, which leads to the proliferation inhibition or death of the cells. Control the power of NIR laser irradiation, the inhibitory effect of the cell proliferation on the aging.EGFRmAb functional modification obviously improves the effect of AuNRs photothermal inhibition, and the simple NIR laser irradiation and EGFRmAb have no obvious cell proliferation inhibition. Due to AuN Rs is located in the mitochondria of cells. We speculate that the above inhibition of cell proliferation may be caused by AuNRs photothermal induced endogenous apoptosis.
The third part is the mechanism of EGFRmAb/AuNRs photothermal induced apoptosis in Hep-2 cells.
Objective: to control the parameters of NIR laser irradiation, observe the apoptosis of Hep-2 cells induced by EGFRmAb/AuNRs photothermal, and to explain the molecular mechanism of apoptosis by detecting the expression of apoptosis related protein in mitochondrial pathway, and prove that the functional modification of EGFRmAb can effectively improve the effect of AuNRs photothermal induced apoptosis of Hep-2 cells.
Methods: with reference to the second parts of the experiment, the parameters of NIR laser irradiation were controlled: power 3.0W/cm2, time 8min. electron microscope observation of ultrastructural changes of Hep-2 cells and apoptosis and cycle of.TUNEL/PI double staining flow cytometry at different time points after irradiation, and then flow cytometry was used to detect Hep-2 cells at different time points after irradiation. Activation of Caspase-3, Bcl-2/Bax and Cyt-c positive expression rate, intracellular ROS level and Ca2+ concentration and cell mitochondrial membrane potential (Delta a m) decline rate.
Results: (1) the apoptosis of NIR+AuNRs and NIR+EGFRmAb/AuNRs groups was observed by 48h electron microscope after irradiation. (2) the cells in group NIR+AuNRs and NIR+EGFRmAb/AuNRs showed significant G0/G1 phase block at 24h and 48h after irradiation, and at the same time, the ratio of S phase decreased and showed a time effect relationship. When compared with the AuNRs group, the EGFRmAb/AuNRs group compared with the AuNRs group at 48h. The decrease of the rate was relatively obvious. The apoptosis of P0.01. (3) NIR+AuNRs group and NIR+EGFRmAb/AuNRs group was obvious after irradiation. The apoptosis rate of.NIR+EGFRmAb/AuNRs group cells in.NIR+EGFRmAb/AuNRs group after irradiation (24h, 48h, 72h) was higher than that of NIR+AuNRs group. The apoptotic rate of NIR+EGFRmAb/AuNRs group at 72h was the highest, up to 73 after P0.05. irradiation, reaching 73. After.63%. (4) irradiation, the cells in group NIR+AuNRs and group NIR+EGFRmAb/AuNRs activated Caspase-3, the positive expression rate of Bax, the decreasing rate of M and the ROS level in the cells, the increase of Ca2+ concentration and the decrease of the positive expression rate of the cells. The change trend of the above indexes was obvious with the time lapse, but the changes of the above indexes in the NIR+ EGFRmAb/AuNRs group were more than those of the NIR+AuNRs group. The positive expression rate of Cyt-c in the cells of group NIR+AuNRs and NIR+EGFRmAb/AuNRs was obviously increased after P0.05. irradiation. The positive expression of Cyt-c was detected at 1h after irradiation, and the highest in 24h and then gradually decreased. The Cyt-c positive expression rate of the NIR+EGFRmAb/AuNRs group was more obvious than that of the NIR+AuNRs group at 24h and 48h after irradiation.
Conclusion: the control of appropriate NIR laser irradiation parameters can induce apoptosis of Hep-2 cells by AuNRs photothermal effect. This apoptosis and cell cycle arrest are closely related to the increase of.ROS and Ca2+ content, delta m decline, Cyt-c release, activation of Caspase-3 up regulation and the decrease of Bcl-2/Bax ratio play an important role in the process of apoptosis, can be seen, line The endogenous pathway mediated by particles is an important way of AuNRs photothermal induced apoptosis of Hep-2 cells..EGFRmAb functional modification can improve the effect of apoptosis through the targeting of AuNRs, but the EGFR monoclonal antibody itself does not participate in the apoptosis.
The fourth part of EGFRmAb/AuNRs photothermal therapy for transplanted tumor of laryngeal squamous cell carcinoma in nude mice
Objective: To observe the effect of AuNRs photothermal therapy in the treatment of subcutaneous laryngeal cancer xenografts in nude mice under local injection and systemic intravenous administration, and to detect the apoptosis of tumor cells after treatment.
Methods: a tumor model of subcutaneous laryngocarcinoma in nude mice was established. The tumor body was partially injected with AuNRs, through the whole body of the tail vein to EGFRmAb/AuNRs, the parameters of the NIR laser irradiation and the third part of the experiment in vitro. The local skin temperature changes were measured during the irradiation. The vernier caliper was used to measure the size of the tumor after irradiation. The experiment was completed at the end of the irradiation for 2 weeks to draw the growth of the transplanted tumor. Tumor cells were harvested after nude mice were sacrificed. Apoptosis and necrosis of tumor cells were detected by TUNEL/PI double staining flow cytometry.
Results: the temperature of the skin tended to be stable after the NIR laser irradiation for 3min. The temperature of the AuNRs local injection group increased by 15 degrees C, the temperature of the EGFRmAb/AuNRs intravenous administration group increased by 11 degrees C. The growth of the tumor in the observation group was obviously inhibited, and the growth rate of the observation time points was obviously slowed, but the AuNRs local injection group The tumor suppressor effect was more obvious after irradiation. P0.05. alone EGFRmAb (E3138, Sigma) intravenous administration, low dose NIR laser irradiation and non EGFRmAb modified AuNRs intravenous administration did not produce significant tumor inhibition effect. Flow cytometry detection, AuNRs local injection group and EGFRmAb/AuNRs intravenous administration group irradiation After 2 weeks, the tumor tissue cells showed obvious apoptosis. The apoptosis rate and the necrosis rate of 58.35% and 30.46%.AuNRs local injection groups were significantly higher than that of the EGFRmAb/AuNRs intravenous group, P0.01.
Conclusion: the effect of AuNRs direct injection of light and heat can obviously inhibit the growth of xenograft in nude mice. EGFRmAb/AuNRs intravenous administration can also play a certain therapeutic effect. Flow cytometry shows the existence of photothermal induced apoptosis. The effect of animal experiments on EGFRmAb/ AuNRs target photothermal treatment of tumor brings hope to the tumor. Because of the complexity of the internal environment, it is necessary to further study the safe and effective AuNRs photothermal therapy for deep tumors in vivo.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R739.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 劉愛(ài)紅;孫康寧;李?lèi)?ài)民;;腫瘤熱療機(jī)制與方法的研究進(jìn)展[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2006年11期

2 彭遠(yuǎn)飛;鄭民華;;腫瘤熱療的細(xì)胞分子作用機(jī)制及應(yīng)用進(jìn)展[J];世界華人消化雜志;2007年12期

3 廖遇平;靶向熱消融治療惡性腫瘤的現(xiàn)狀[J];中國(guó)現(xiàn)代醫(yī)學(xué)雜志;2004年03期

，

本文編號(hào)：2110405