【作者】 樊伟；

【导师】 何晓光； 董守安； 李玉晓； 王福科；

【作者基本信息】 昆明医学院 ， 耳鼻咽喉—头颈外科学， 2010， 硕士

【摘要】 [目的]探讨分子靶向性金纳米棒的制备、观察金纳米棒进入细胞的过程及其与抗体共轭前后细胞毒性的变化并检测其与抗体共轭后提高近红外激光热疗的效果,为临床治疗皮肤基底细胞癌提供一种新思路。[方法]体外培养A-431人表皮癌细胞和正常人皮肤成纤维细胞,同时合成金纳米棒并使其与抗表皮生长因子受体单克隆抗体共轭结合后具有分子靶向性。进行以下实验：(1)对制备的金纳米棒进行表征。(2)改良MTT法检测不同浓度的金纳米棒及其与抗体共轭后对两种细胞毒性的变化。(3)金纳米棒与A-431细胞孵育不同时间后透射电镜观察其进入细胞的过程。(4)两种细胞分为四组：①阴性对照组；②单纯照射组；③25%金纳米棒组；④25%抗体／金纳米棒组；②③④组经不同功率近红外激光照射后改良MTT法检测细胞生长活力,台盼蓝染色观察细胞形态学变化。实验所得数据用均数±标准差(X±+S)表示,应用SPSS16.0统计学软件包进行分析,数据采用单因素方差分析进行组间比较,以P<0.05判定差异具有统计学意义。[结果](1)制备的金纳米颗粒呈棒状,平均粒径为35-45nm,光密度值为0.9,在540nm和822nm附近有两个等离子共振吸收峰,其822nm附近的吸收峰处于近红外区域(800-1200nm)是肌体组织的安全透射窗口,则其可用于近红外热疗。(2)改良MTT法检测细胞毒性时发现,浓度为25%以下金纳米棒和50%以下抗体／金纳米棒共轭物为两种细胞的安全浓度。(3)金纳米棒与A-431细胞共同孵育1h后金纳米棒主要存在细胞外,4h后细胞膜伸出伪足包裹吞噬金纳米棒,8-24h内在细胞质内可见大量包裹着金纳米棒的吞噬泡,细胞核内未见到金纳米棒,48h后金纳米棒又大量出现在细胞外。(4)两种细胞分别与25%的金纳米棒和抗体／金纳米棒共轭物培养后用不同功率近红外激光进行热疗,改良MTT法检测细胞活力,根据公式计算细胞相对存活率；同时台盼蓝染色观察细胞形态变化。A-431细胞：②③④组经相同激光功率照射后,④组细胞相对存活率显著低于②③组(P值均<0.01),③组细胞相对存活率也显著低于②组(P值<0.05),台盼蓝染色后②③④组间比较也发现：④组杀灭大量的细胞所用激光功率远小于②③组(P值均<0.01),③组杀灭大量的细胞所用激光功率也远小于②组(P值<0.05)。比较正常成纤维细胞的②③④组时得到结果与A-431细胞的相似,但是③④组数据组间比较(P值>0.05)。A-431细胞和正常成纤维细胞两种细胞②③④组各组间两两比较发现：相同激光功率照射后②③④组中的A-431细胞的相对细胞存活率均远小于正常成纤维细胞②③④组(P值均<0.05)。[结论]：1、采用光化学法合成了金纳米棒。金纳米棒平均粒径为35-45nm,光密度值为0.9,其纵向吸收峰位于822nm附近属于近红外区域(800-1200nm),可用于近红外激光热疗。2、制备成的抗表皮生长因子受体单克隆抗体／金纳米棒共轭物在细胞内的安全浓度远高于金纳米棒,提高了其生物相容性。同时也增加了金纳米棒的稳定性和分散性,使得其在生物医学领域有着更为广阔的应用前景。3、金纳米棒能以纳米颗粒的形式,以细胞吞噬的方式大量进入A-431细胞内,进入细胞内的金纳米棒形状也未遭到破坏。4、金纳米棒与抗表皮生长因子受体单克隆抗体共轭后对A-431细胞具有较好的分子靶向性,应用其进行近红外激光热疗时,与单纯金纳米棒相比,抗体／金纳米棒共轭物只需14w/cm~2的近红外激光照射后对A-431人皮肤癌细胞生长抑制率即可达87%以上,而用此激光功率照射正常人成纤维细胞时对其的生长抑制作用很小。5、近红外激光热疗后,A-431人皮肤癌细胞比正常人成纤维细胞对热更敏感。更多还原

【Abstract】 [Objective] Investigate the preparation of molecular targeted gold nanorods, observation of gold nanorods into the process of cell, conjugated of gold nanorods before and after the change of cell toxicity and detection of the conjugated gold nanorods to improve the effect of infrared hyperthermia, for the treatment of basal cell carcinoma provides a new way.[Methods] Cultured A-431 human epidermal cancer cells and normal human skin fibroblasts in vitro, at the same time synthesis of gold nanorods and also to conjugated anti-epidermal growth factor receptor monoclonal antibody with molecular targeted. Conducted the following experiment:(1)Observe the characteristics of gold nanorods was prepared. (2) Improved MTT Colorimetric assay with different concentrations of gold nanorods and gold nanorods conjugated the toxicity of the two cells. (3)Gold bars and A-431 cells were incubated for different time transmission electron microscopy to observe the process of entering cells. (4) Divided into four groups of two cells:①egative control group,②aser irradiation alone,③25% concentrations of gold nanorods,④25% concentrations of anti-EGFR/ Au nanorods,②③④By different power of infrared laser photo-thermal therapy, then Modified MTT assay cell viability, cell survival rate according to formula, Trypan blue staining observed changes in cell morphology. Experimental data obtained with (x±s). Express, Application of statistical package with SPSS 16.0 statistical analysis, Data were statistically analyzed using a one-way analysis of variance, P<0.05 determined statistical significance. [Results] (1) Preparation of gold nanoparticles are bat, Average diameter is 35-45nm, Optical density around 0.8, there were two plasma resonance absorption peak in the vicinity of 520nm and 820nm. The 820nm absorption peak in the vicinity of near-infrared region (800-1200nm) is the organization’s security body the transmission window. It can be used for near infrared laser thermotherapy. (2) Improved MTT Colorimetric assay cell-cytotoxic:Concentration of less than 25% of the gold nanorods and concentration of less than 50% of the anti-EGFR/Au nanorods were the safe concentration for the two cell. (3) Gold nanorods and A-431 cells were incubated after 1h, gold nanorods was also found primarily in extracellular.4h after the cell membrane out of swallowing gold nanorods wrapped pseudopodia.8-24h within the cytoplasm shows a large number of gold nanorods wrapped in bubble phagocytosis, gold nanorods are not seen in the cell nucleus.48h after the large number of gold nanorods have appeared in the extracellular. (4) Two kinds of cells respectively and 25% of the concentration of gold nanorods and 25% of the concentration of the anti-EGFR/Au nanorods incubated, then different power of infrared laser photo-thermal therapy. Improved MTT Colorimetric assay the cell growth activity, Trypan blue staining observed changes in cell morphology. A-431 cell:②③④roup irradiated by the same laser power,④roup cells relative survival rate was significantly lower than②③group(P< 0.05). Also③group cells relative survival rate was significantly lower than②roup(P<0.05). Trypan blue staining②③④group found:④roup a large number of cells were killed need a laser power is much smaller than②③group(P<0.05).③roup to kill a large number of cells used in laser power is also far less than②roup. Comparison of normal fibroblast②③④roup can get the same results. But the comparison group data③④(P>0.05). Comparison of A-431 cells and normal fibroblasts of the two cell②③④group found:After the same laser power irradiation, group②③④the A-431 cells of the relative survival rat are much smaller than normal fibroblasts(P<0.05).[Conclusions]1. Photochemically synthesized gold nanorods. Average diameter is 35-45nm, Optical density around 0.9. The 822nm absorption peak in the vicinity of near-infrared region (800-1200nm) is the organization’s security body the transmission window. It can be used for near infrared laser thermotherapy. 2. Prepared into the anti-epidermal growth factor receptor monoclonal antibody/ conjugated gold nanorods in the cell body of the safe concentration is much higher than pure gold nanorods. Enhance its biocompatibility. At the same time combined with the antibody also increased the stability of gold nanorods and dispersion, Making it in the biomedical field has more potential applications.3. Gold nanorods can form nanoparticles and phagocytosis way can a large number of gold nanorods entering A-431 cells. Into the cells shape of gold nanorods have not been destroyed.4. Anti-EGFR/Au nanorods prepared by a combination with a high molecular target-specific to the A-431 cells, Compared with pure gold nanorods, Anti-EGFR/Au nanorods just 14w/cm~2 near-infrared laser irradiation on the A-431 of human skin cancer cell growth inhibition rate of 87% or more can. While the laser power irradiation on the growth of normal fibroblasts very little inhibition.5. After infrared laser photo-thermal therapy, A-431 cells than normal human skin fibroblasts more sensitive to heat.更多还原