功能化修饰金纳米棒光热作用诱发喉鳞癌细胞凋亡机制研究

发布时间：2018-03-13 10:16

本文选题：金纳米棒　切入点：光热作用　出处：《昆明医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：[目的]细胞可以通过自噬维持生物形式的稳态,而自噬则依赖于溶酶体的参与才得以完成。金纳米微粒成为纳米生物技术研究领域里的典型代表,较多地应用在肿瘤的诊断和治疗方面。因此,本研究采用西妥昔单抗作功能化修饰物、以Hep-2喉鳞癌细胞系作为研究对象、构建EGFRmAb/AuNRs靶向聚集癌细胞的理想模型,期望获得协同增强金纳米棒光热治疗肿瘤的效果,为进一步研究探索自噬/溶酶体途径在IMC-C225 /AuNRs光热诱导喉鳞癌细胞程序性死亡中的调控机制奠定实验基础。[方法]1.倒置显微镜观察细胞形态;2.金纳米棒光热升温实验;3.运用MTT检测细胞增殖活性;4. Western Blot检测蛋白水平变化5. RT-qPCR检测细胞内mRNA水平变化;6.电子透射显微镜观察自噬体。[结果]1. AuNRs与Hep-2细胞共培养显微镜下观察细胞形态,AuNRs/Hep-2共培养的细胞形态较正常Hep-2的无明显变化。MTT法分析细胞1-7天生长情况结果示:两种细胞大致呈“S”型曲线生长,AuNRs并没有表现出明显的细胞毒性。2.不同浓度AuNRs光热作用升温实验AuNRs经NIR照射后确实存在很明显的光热效应(最高达36.5℃)并在3min时能达到一个特定的温度并保持恒定,这与文献一致(图3b)。所以我们以后的实验照射时间选定为5min。3.不同浓度AuNRs搭配NIR照射对细胞活力的影响在0nmol/L、0.3nmol/L、0.6nmol/L、0.9nmol/LHep-2/AuNRs 四组中,NNIR组细胞存活率分别为100%、98. 47%、96.60%、97.17%; YNIR组细胞存活率分别为 55. 56%、39.69%、35.39%、16.21%,进一步比较 NNIR 组和 YNIR组细胞存活率,p0. 01,差异有统计学意义,说明NIR照射对细胞有显著的杀伤效果。4. IMC-C225/AuNRs搭配NIR照射对细胞活力的影响在5W、6W、7W、8W 照射强度下,Hep-2+0. 3nmol/L AuNRs 组Hep-2+IMC-C225/AuNRs 组的细胞存活率分别为 87. 37%、86. 88%、84. 95%、83. 25% 和 95. 20%、74. 76%、54.49%、24.45%,其中 5W 组 p 值无意义,6W组p0. 05, 7W组p0. 01,8W组p0.01,差异均有统计学意义,并随着AuNRs浓度的升高Hep-2细胞的抑制率也升高。结果示:IMC-C225/AuNRs,即使在较弱的NIR照射条件下,出现了明显的细胞抑制效果,对癌细胞有极强的杀伤力。5.加入溶酶体自噬抑制剂后,IMC-C225/AuNRs搭配NIR照对细胞活力的影响YNIR 条件下,Hep-2+3-MA+CQ+IMC/AuNRs 组和 Hep-2+CA-074+PA+IMC/AuNRs组的细胞存活率分别为46. 19%和50. 44%,两组间比较p0. 01,差异有统计学意义。结果示:加入溶酶体自噬抑制剂后阻断了溶酶体自噬对癌细胞的保护作用,从而进一步增强IMC-AuNRs对癌细胞的杀伤力。6. Western Blot检测蛋白水平变化YNIR组自噬相关蛋白LC3II明显表达,并随着金纳米棒浓度升高而升高;YNIR组与NIR组相比,p0.05,差异均有统计学意义。7. RT-qPCR检测细胞内mRNA水平变化所选取的6个自噬相关基因ATG13、LC3、P62、ATG5、ULK1、VPS34D的mRNA均会随着纳米金棒浓度升高被抑制,并且YNIR组自噬相关基因mRNA的表达下降的更为明显;NNIR组与YNIR组相比p0. 05,差异有统计学意义。8.电子透射显微镜观察自噬体形成各组细胞状态良好,无明显凋亡现象,部分细胞有自噬现象出现(发现双层的自噬溶酶体)。Ⅱ组和Ⅲ组的少部分细胞中可以观察到长条型的固体物质(长径不到100nmol/L的黑色物质)集中富集在线粒体或溶酶体中。而且在相同放大倍数下,Ⅲ组中富集该固体物质的细胞数量明显多于Ⅱ组。[结论]1.不同浓度AuNRs经NIR照射后存在很明显的光热效应并对Hep-2细胞有显著杀伤作用。2. IMC-C225/AuNRs的金纳米棒即使在较弱的NIR照射后产生了明显的细胞抑制率。3.加入溶酶体自噬抑制剂,阻断了溶酶体自噬对癌细胞的保护作用,从而进一步增强IMC-C225/AuNRs对癌细胞的杀伤力。4.自噬参与了功能化修饰金纳米棒光热作用诱导喉鳞癌细胞死亡的过程。
[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.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.65

【参考文献】