本文選題：黑素瘤 + RGD多肽　； 參考：《安徽醫(yī)科大學》2010年博士論文

【摘要】： 背景早期診斷和治療是腫瘤研究中的關鍵科學問題。納米科技的快速發(fā)展為解決這一關鍵科學問題帶來了新機遇。具有獨特等離子共振效應的金納米棒粒子有潛力應用于腫瘤的早期診斷和治療。 目的本研究將利用樹形分子取代金納米棒表面的毒性分子十六烷基三乙基溴化銨(CTAB),然后與精氨酸-甘氨酸-天冬氨酸多肽(RGD)偶聯(lián)構建生物相容性好的靶向腫瘤細胞和腫瘤血管內皮細胞的生物探針,并解決在病變局部選擇性治療的理論問題,旨在為黑素瘤的早期診斷和治療提供新的策略。 方法首先應用“晶種生長”法合成均一的金納米棒粒子(GNRs),然后利用巰基改性的G4.0聚酰胺胺(PAMAM)樹形分子替代金納米棒表面的CTAB分子。PAMAM樹形分子修飾的納米粒子與生物配體RGD多肽通過共價鍵偶聯(lián)得到具有靶向功能的生物探針(RGD-dGNRs)。利用高分辨率透射電鏡、原子力顯微鏡、核磁共振波譜和紫外-可見吸收光譜等儀器對納米探針進行表征。 選擇人臍靜脈血管內皮細胞(HUVEC)、黑素瘤A375和乳腺癌MCF-7等細胞系來研究納米探針的靶向性。應用CCK-8方法評價RGD-dGNRs納米探針對細胞的毒性作用。利用暗視野顯微鏡來觀察RGD-dGNRs納米探針與腫瘤細胞結合的敏感性和特異性。同時設立嚴格的競爭抑制結合實驗來推測細胞的結合位點。當腫瘤細胞與RGD-dGNRs納米探針結合后,應用與金納米棒縱向等離子共振吸收峰相近的近紅外(NIR)808nm激光照射,能量分別采用30 mW,70 mW,110 mW,150 mW四個等級,光斑直徑為1.0 mm,照射時間為4分鐘。應用Trypan blue(臺盼藍)染色和Calcein-AM染色評價納米探針吸收激光轉化為熱能后殺傷腫瘤細胞的效能。 將5×106黑素瘤細胞注射到裸鼠右側大腿皮下,當腫瘤直徑達到5mm左右,200μg的RGD-dGNRs納米探針從尾靜脈注入小鼠體內,然后分別在3h, 6h, 9h和12h四個時間點處死3只小鼠,收集血液、腫瘤組織以及心、肝、腎等重要臟器,用電感偶合質譜儀(ICP-Mass)測定各個臟器和腫瘤組織的金納米棒含量,評價RGD-dGNRs納米探針在動物體內的分布狀況。為推測RGD-dGNRs納米探針與細胞結合的位點,同時進行競爭抑制結合實驗,在注射RGD-dGNRs納米探針前,給予小鼠過量的RGD多肽(0.5mg)。 30只荷瘤小鼠被隨機分為三組,分別為實驗組(200μg RGD-dGNRs +NIR激光照射);PBS對照組(PBS+NIR激光照射);空白對照組(未予以任何處理)。納米探針從小鼠靜脈注射6小時后,腫瘤部位予以NIR的808nm激光照射,功率密度24 W/cm2,光斑直徑5mm,照射時間5分鐘。每周治療1次,共治療4次。應用皮膚反射式共聚焦顯微鏡(RCM)觀察腫瘤的組織學和血流變化。通過觀察腫瘤體積變化和Kaplan-Meier生存曲線來觀察RGD-dGNRs光熱治療對小鼠生存期的影響。結果通過“晶種生長法”制備的金納米棒(GNRs-CTAB)大小均一,面徑比為4.2左右,橫向等離子共振吸收峰在520 nm,縱向等離子共振吸收峰在821nm左右。樹形分子修飾后的金納米棒(dGNRs)顯示了很好的分散性。RGD-dGNRs納米探針的縱向等離子共振吸收峰發(fā)生了輕度紅移(3nm)。RGD-dGNRs納米探針在生理環(huán)境下顯示了很好的分散性和穩(wěn)定性。金納米棒探針在200ug/ml濃度范圍內未見明顯的細胞毒性作用,而GNRs-CTAB在濃度在50μg/ml左右即可顯示明顯的細胞毒性作用。 在暗視野顯微鏡觀察下,A375黑素瘤細胞和HUVEC細胞與RGD-dGNRs納米探針結合后顯示明亮的金黃色,不能被水沖洗掉。過量的RGD多肽明顯抑制了RGD-dGNRs納米探針與黑素瘤細胞的結合。另外RGD-dGNRs納米探針并不與αvβ3整合素陰性乳腺癌細MCF-7胞結合。 A375黑素瘤細胞與RGD-dGNR納米探針結合30分鐘后,在70mW的NIR激光照射下,可見部分細胞被殺死,當激光能量達到110mW時,細胞死亡明顯增加。dGNRs納米粒子孵育或單純的NIR激光照射并未導致細胞明顯死亡。RGD-dGNRs納米探針與αVβ3整合素陰性的MCF-7乳腺癌細胞孵育,然后在NIR激光照射下,未見明顯的細胞死亡。細胞的殺傷情況與RGD-dGNR納米探針的濃度也密切相關。在110m的激光能量照射下,當RGD-dGNR納米探針的濃度增加到100ug/ml時,幾乎所有的黑素瘤細胞被殺死。因此RGD-dGNRs體外光熱治療的最佳濃度可能為100μg/ml。 藥代動力學實驗顯示,隨著時間的延長,RGD-dGNRs納米探針在腫瘤部位的蓄積逐漸增加。6小時后,RGD-dGNRs納米探針在腫瘤部位可見明顯的蓄積,約占金納米棒總量的17%。過量的RGD多肽幾乎完全抑制了RGD-dGNRs納米探針在腫瘤部位的蓄積。與空白對照組和PBS治療組相比,實驗組小鼠的腫瘤體積在整個觀察期間明顯變小(P㩳0.01),其中四只小鼠的腫瘤組織幾乎完全消失�？瞻讓φ战M小鼠的平均生存期大約為3周,PBS治療組的小鼠平均生存期大約為4周,而實驗組小鼠的平均生存期大于7周,組間有顯著性差異(P =0.006)。Kaplan-Meier曲線也顯示RGD-dGNRs納米探針的光熱治療能明顯延長小鼠的生存時間。 RCM實時觀察到RGD-dGNRs光熱治療荷瘤小鼠3小時后,腫瘤部位血流明顯減少,而對照組小鼠的腫瘤組織血流無明顯改變。治療7周后,RCM觀察到RGD-dGNRs納米探針光熱治療小鼠的腫瘤組織內出現(xiàn)大量的網(wǎng)狀或樹枝狀膠原纖維束,組織學HE染色顯示為大量膠原纖維增生,類似疤痕結構。而RCM在對照組小鼠的腫瘤組織內可見大量折射增強的細胞,部分細胞聚集成簇,其對應的HE染色可見大量的黑素瘤細胞。 結論本研究成功合成了生物相容好的RGD-dGNRs納米探針,這些新穎的納米探針能準確識別活體腫瘤細胞與腫瘤中的新生血管內皮細胞。在NIR激光照射下,納米探針吸收光能轉化為熱能將腫瘤血管破壞和腫瘤細胞殺傷。這種納米復合物探針也適用于其他αvβ3整合素陽性的腫瘤細胞。這些智能化的生物探針在未來的腫瘤分子成像與光熱治療方面具有潛在的應用價值。
[Abstract]:Background Early diagnosis and treatment are key scientific problems in tumor research . The rapid development of nanotechnology brings new opportunities to solve this critical scientific problem . The gold nanorod particles with unique plasma resonance effects have the potential to be applied to early diagnosis and treatment of tumors .



Objective In order to provide a new strategy for the early diagnosis and treatment of melanoma , the cytotoxic molecule cetyl trimethyl ammonium bromide ( CTAB ) of gold nanorod surface was replaced with tree - shaped molecule , and then coupled with the arginine - glycine - aspartic acid polypeptide ( RGD ) to construct a biological probe for targeting tumor cells and tumor vascular endothelial cells .



Methods All gold nanorod particles ( GNRs ) were synthesized by " seed growth " method , and then the CTAB molecules on the surface of gold nanorods were replaced by thiol - modified G4 .



The sensitivity and specificity of RGD - dGNRs nano - probe to tumor cells were studied by using CCK - 8 method . The sensitivity and specificity of RGD - dGNRs probe to tumor cells were evaluated by CCK - 8 method .



5 脳 106 melanoma cells were injected subcutaneously into the right thigh of nude mice . When the diameter of tumor reached 5 mm , 200 渭g of RGD - dGNRs nano - probe was injected from the tail vein into the mice . Then , three mice were sacrificed at 3 h , 6 h , 9 h and 12 h . The distribution of RGD - dGNRs probe was evaluated .



Thirty - six tumor - bearing mice were randomly divided into three groups : experimental group ( 200 渭g RGD - dGNRs + NIR laser irradiation ) , PBS control group ( PBS + NIR laser irradiation ) , PBS control group ( PBS + NIR laser irradiation ) .



Under a dark field microscope , A375 melanoma cells and HUVEC cells were combined with RGD - dGNRs nanoprobes to show bright golden yellow , which could not be washed away by water . Excess RGD polypeptide significantly inhibited the binding of RGD - dGNRs nanoprobes to melanoma cells . The RGD - dGNRs nanoprobes did not bind to 偽v尾3 integrin negative breast cancer cells MCF - 7 .



After 30 minutes of binding of A375 melanoma cells with RGD - dGNR nano - probe , some cells were killed when the laser energy reached 110 mW . The cell death was significantly increased when the laser energy reached 110 mW . The cell killing conditions were also closely related to the concentration of RGD - dGNR nano - probe .



In comparison with the control group and PBS group , the tumor volume of the RGD - dGNRs nano - probe was significantly reduced ( P ? 0.01 ) . The average survival time of the mice in the experimental group was about 4 weeks compared with the blank control group and the PBS treatment group , while the average survival time of the mice in the experimental group was more than 7 weeks , and there was a significant difference between the groups ( P = 0.006 ) . Kaplan - Meier curves also show that the photothermal treatment of RGD - dGNRs nanoprobes can significantly prolong the survival time of mice .



After 7 weeks of treatment , a large number of reticular or dendritic collagen bundles appeared in the tumor tissues of mice with RGD - dGNRs .



Conclusion The biological compatible RGD - dGNRs nanoprobes are successfully synthesized in this study . These novel nano - probes can accurately identify tumor cells and tumor cells in vivo .

【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R739.5

【引證文獻】

相關博士學位論文 前3條

1 張世文;EGFRmAb功能化修飾金納米棒光熱誘導喉鱗癌細胞凋亡分子機制研究[D];昆明醫(yī)科大學;2012年

2 黃鵬;納米材料的可控合成、功能化及其在腫瘤診療中應用研究[D];上海交通大學;2012年

3 李西波;載阿霉素葡聚糖金磁復合微粒在肝癌磁導靶向治療中的應用研究[D];西北大學;2013年

相關碩士學位論文 前1條

1 劉芮灤;基于ARM的LED光熱治療系統(tǒng)設計與初步實驗研究[D];重慶大學;2012年

，

本文編號：1870971