本文選題：金納米棒　切入點：光熱作用　出處：《昆明醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：[目的]細(xì)胞可以通過自噬維持生物形式的穩(wěn)態(tài),而自噬則依賴于溶酶體的參與才得以完成。金納米微粒成為納米生物技術(shù)研究領(lǐng)域里的典型代表,較多地應(yīng)用在腫瘤的診斷和治療方面。因此,本研究采用西妥昔單抗作功能化修飾物、以Hep-2喉鱗癌細(xì)胞系作為研究對象、構(gòu)建EGFRmAb/AuNRs靶向聚集癌細(xì)胞的理想模型,期望獲得協(xié)同增強金納米棒光熱治療腫瘤的效果,為進(jìn)一步研究探索自噬/溶酶體途徑在IMC-C225 /AuNRs光熱誘導(dǎo)喉鱗癌細(xì)胞程序性死亡中的調(diào)控機制奠定實驗基礎(chǔ)。[方法]1.倒置顯微鏡觀察細(xì)胞形態(tài);2.金納米棒光熱升溫實驗;3.運用MTT檢測細(xì)胞增殖活性;4. Western Blot檢測蛋白水平變化5. RT-qPCR檢測細(xì)胞內(nèi)mRNA水平變化;6.電子透射顯微鏡觀察自噬體。[結(jié)果]1. AuNRs與Hep-2細(xì)胞共培養(yǎng)顯微鏡下觀察細(xì)胞形態(tài),AuNRs/Hep-2共培養(yǎng)的細(xì)胞形態(tài)較正常Hep-2的無明顯變化。MTT法分析細(xì)胞1-7天生長情況結(jié)果示:兩種細(xì)胞大致呈“S”型曲線生長,AuNRs并沒有表現(xiàn)出明顯的細(xì)胞毒性。2.不同濃度AuNRs光熱作用升溫實驗AuNRs經(jīng)NIR照射后確實存在很明顯的光熱效應(yīng)(最高達(dá)36.5℃)并在3min時能達(dá)到一個特定的溫度并保持恒定,這與文獻(xiàn)一致(圖3b)。所以我們以后的實驗照射時間選定為5min。3.不同濃度AuNRs搭配NIR照射對細(xì)胞活力的影響在0nmol/L、0.3nmol/L、0.6nmol/L、0.9nmol/LHep-2/AuNRs 四組中,NNIR組細(xì)胞存活率分別為100%、98. 47%、96.60%、97.17%; YNIR組細(xì)胞存活率分別為 55. 56%、39.69%、35.39%、16.21%,進(jìn)一步比較 NNIR 組和 YNIR組細(xì)胞存活率,p0. 01,差異有統(tǒng)計學(xué)意義,說明NIR照射對細(xì)胞有顯著的殺傷效果。4. IMC-C225/AuNRs搭配NIR照射對細(xì)胞活力的影響在5W、6W、7W、8W 照射強度下,Hep-2+0. 3nmol/L AuNRs 組Hep-2+IMC-C225/AuNRs 組的細(xì)胞存活率分別為 87. 37%、86. 88%、84. 95%、83. 25% 和 95. 20%、74. 76%、54.49%、24.45%,其中 5W 組 p 值無意義,6W組p0. 05, 7W組p0. 01,8W組p0.01,差異均有統(tǒng)計學(xué)意義,并隨著AuNRs濃度的升高Hep-2細(xì)胞的抑制率也升高。結(jié)果示:IMC-C225/AuNRs,即使在較弱的NIR照射條件下,出現(xiàn)了明顯的細(xì)胞抑制效果,對癌細(xì)胞有極強的殺傷力。5.加入溶酶體自噬抑制劑后,IMC-C225/AuNRs搭配NIR照對細(xì)胞活力的影響YNIR 條件下,Hep-2+3-MA+CQ+IMC/AuNRs 組和 Hep-2+CA-074+PA+IMC/AuNRs組的細(xì)胞存活率分別為46. 19%和50. 44%,兩組間比較p0. 01,差異有統(tǒng)計學(xué)意義。結(jié)果示:加入溶酶體自噬抑制劑后阻斷了溶酶體自噬對癌細(xì)胞的保護作用,從而進(jìn)一步增強IMC-AuNRs對癌細(xì)胞的殺傷力。6. Western Blot檢測蛋白水平變化YNIR組自噬相關(guān)蛋白LC3II明顯表達(dá),并隨著金納米棒濃度升高而升高;YNIR組與NIR組相比,p0.05,差異均有統(tǒng)計學(xué)意義。7. RT-qPCR檢測細(xì)胞內(nèi)mRNA水平變化所選取的6個自噬相關(guān)基因ATG13、LC3、P62、ATG5、ULK1、VPS34D的mRNA均會隨著納米金棒濃度升高被抑制,并且YNIR組自噬相關(guān)基因mRNA的表達(dá)下降的更為明顯;NNIR組與YNIR組相比p0. 05,差異有統(tǒng)計學(xué)意義。8.電子透射顯微鏡觀察自噬體形成各組細(xì)胞狀態(tài)良好,無明顯凋亡現(xiàn)象,部分細(xì)胞有自噬現(xiàn)象出現(xiàn)(發(fā)現(xiàn)雙層的自噬溶酶體)。Ⅱ組和Ⅲ組的少部分細(xì)胞中可以觀察到長條型的固體物質(zhì)(長徑不到100nmol/L的黑色物質(zhì))集中富集在線粒體或溶酶體中。而且在相同放大倍數(shù)下,Ⅲ組中富集該固體物質(zhì)的細(xì)胞數(shù)量明顯多于Ⅱ組。[結(jié)論]1.不同濃度AuNRs經(jīng)NIR照射后存在很明顯的光熱效應(yīng)并對Hep-2細(xì)胞有顯著殺傷作用。2. IMC-C225/AuNRs的金納米棒即使在較弱的NIR照射后產(chǎn)生了明顯的細(xì)胞抑制率。3.加入溶酶體自噬抑制劑,阻斷了溶酶體自噬對癌細(xì)胞的保護作用,從而進(jìn)一步增強IMC-C225/AuNRs對癌細(xì)胞的殺傷力。4.自噬參與了功能化修飾金納米棒光熱作用誘導(dǎo)喉鱗癌細(xì)胞死亡的過程。
[Abstract]:[Objective] to maintain steady state in the form of biological cells through autophagy, autophagy is dependent on lysosomal involvement to complete. Gold nanoparticles as a representative of the research field of nano biotechnology, widely used in the diagnosis and treatment of tumor. Therefore, this study adopts cetuximab for functional modification in Hep-2, laryngeal squamous cell carcinoma cell lines as the research object, the construction of EGFRmAb/AuNRs targeting the ideal model aggregation of cancer cells, expect a synergistic enhancement effect of gold nanorods photothermal therapy of tumor, cell morphology was observed. The experimental method for]1. based inverted microscope to further explore the regulation mechanism of autophagy / lysosomal pathway in IMC-C225 induced by /AuNRs thermal throat programmed cell death in squamous cell carcinoma; 2. gold nanorods using photothermal heating experiment; 3. MTT was used to detect cell proliferation activity; protein level detection of 4. Western Blot Changes of mRNA levels of 5. RT-qPCR were detected in 6.; transmission electron microscopy observation of autophagosomes. Results]1. AuNRs Hep-2 cells were co cultured with microscope, cells were analyzed 1-7 days growth results showed no significant changes in.MTT co culture AuNRs/Hep-2 cells than normal Hep-2 two cells is roughly "S" the growth curve, AuNRs and AuNRs showed no obvious cytotoxicity of the photothermal effect of different concentrations of.2. heating experiment of AuNRs irradiated by NIR the existence of photothermal effect obviously (up to 36.5 DEG C) and at 3min can reach a specific temperature and keep constant, which consistent with the literature (Figure 3B) the irradiation time. So we selected 5min.3. of different concentration AuNRs collocation effects of NIR irradiation on cell viability in 0nmol/L, 0.3nmol/L, 0.6nmol/L, 0.9nmol/LHep-2/AuNRs four group, NNIR group of cells The survival rates were 100%, 98.47%, 96.60%, 97.17%; the survival rate in YNIR group were 55.56%, 39.69%, 35.39%, 16.21%, further comparison of NNIR group and YNIR group, the cell survival rate, p0. 01, the difference was statistically significant, indicating that NIR irradiation has significant cytotoxic effect of.4. IMC-C225/AuNRs collocation effects of NIR irradiation on cell viability in 5W, 7W, 8W on cell 6W, irradiation intensity, Hep-2+0. 3nmol/L AuNRs cell group, the survival rate of Hep-2+IMC-C225/AuNRs group were 87.37%, 86.88%, 84.95%, 83.25% and 95.20%, 74.76%, 54.49%, 24.45%, in the 5W group P value is meaningless, 6W group p0. 05, group 7W p0. group 01,8W P0.01. The differences were statistically significant, and with the increase of Hep-2 cell inhibition rate also increased with AuNRs concentration. The results showed: IMC-C225/AuNRs, NIR irradiation even in mild conditions, there is an obvious inhibition effect, strong on cancer cells .5. joined the lysosomal autophagy inhibitor lethality, IMC-C225/AuNRs NIR collocation illumination conditions of YNIR on cell viability, Hep-2+3-MA+CQ+IMC/AuNRs group and Hep-2+CA-074+PA+IMC/AuNRs group, the cell survival rate were 46.19% and 50.44%, between the two groups of p0. 01, the difference was statistically significant. The results showed: adding lysosomal autophagy inhibitor blocked the protective effect of autophagy lysosome after in cancer cells, so as to enhance the effect of IMC-AuNRs on the protein level detection of.6. Western Blot YNIR group changed the lethality of autophagy related protein LC3II expression, and increased with the concentration of gold nanorods increased; compared with the YNIR group, NIR group and P0.05, 6 of autophagy related gene ATG13, mRNA level there was a significant difference in.7. RT-qPCR detection of intracellular selected LC3, P62, ATG5, ULK1, VPS34D mRNA with gold nanorods concentration was inhibited System, and the expression of mRNA gene in YNIR group decreased autophagy was more obvious; NNIR group and YNIR group compared to p0. 05, there were statistically significant differences in.8. transmission electron microscopy to observe the autophagosome formation state of cells were good, no obvious apoptosis phenomenon, some cell autophagy phenomenon (found autolysosome double) a few. The cells of group II and group III were observed in the solid material strip type (length to black substance 100nmol/L) concentration in mitochondria and lysosomes. And at the same magnification, the number of cells in group III enrichment of solid material was significantly more than group II. Conclusion]1. with different concentration of AuNRs by NIR after irradiation there photothermal effect obviously and has significant killing effect of.2. IMC-C225/AuNRs gold nanorods even in weak NIR after irradiation have obvious inhibition rate of.3. in solution on Hep-2 cells The inhibition of lysosomal autophagy on cancer cells is inhibited by autophagy inhibitor, which further enhances the killing effect of IMC-C225/AuNRs on cancer cells..4. autophagy is involved in the process of functionalized gold nanorods photothermal induction of laryngeal squamous cell carcinoma.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.65

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