硫化铜微粒在消化道肿瘤光热治疗中的应用研究

发布时间：2018-05-02 22:58

本文选题：硫化铜 + 光热治疗　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:消化道肿瘤呈全球高发趋势,威胁人类的健康,造成巨大的社会医疗经济负担。目前,早诊早治是防治消化道肿瘤的有效方式,结合快速发展纳米探针与分子影像技术实现肿瘤诊疗一体化,是提高肿瘤检出率、提高治疗效果的有效方法。近年来,光热治疗(Photothermal therapy,PTT)已成为一种肿瘤诊疗一体化新模式,展现了巨大的转化医学和临床应用潜力。近期研究表明硫化铜具有高效的光热转化效能,同时具有高信噪比的光声成像对比度,是一种可以实现光声成像-光热治疗的诊疗一体化介质。本研究通过离体测定亚微米级硫化铜理化性质、980nm近红外激光照射Cu S颗粒光热转化效率评估亚微米级硫化铜光热效应,通过细胞水平和活体动物水平实验分析硫化铜光热治疗效果、生物毒性和安全性,为硫化铜的临床转化应用探寻新的方向与应用模式,为肿瘤诊疗一体化探索新的技术。方法:利用四种不同粒径范围的亚微米级硫化铜粉末进行实验探究。这四种亚微米级Cu S粒径范围分别约为80-100nm,100-200nm,200-400nm,300-600nm。实验内容包括亚微米级硫化铜的性质测定、体外细胞实验以及小动物活体实验三个部分。1、电镜观察Cu S粒径范围,测定不同粒径Cu S光热转换性质,观察浓度梯度以及激光照射时间对光热转换温度的影响;测定Cu S微粒光热稳定性,观察多次照射后光热转换效率变化情况。2、显微镜体外观察将Cu S亚微米颗粒与结肠癌细胞孵育后对活细胞生长的影响,利用CCK-8法观察四种粒径、不同浓度的Cu S溶液对结肠癌细胞的细胞毒性。用980nm近红外激光照射含不同浓度Cu S颗粒的细胞培养基10min,观察培养基温度与Cu S浓度梯度、照射时间的关系。利用Annexin V-FITC/PI对近红外激光照射前后的细胞凋亡情况进行分析,研究Cu S光热效应对的杀伤作用。3、利用GFP-Luc SW480结肠癌细胞株制备裸鼠皮下移植瘤,肿瘤长至7mm左右时进行小动物活体实验,通过小动物活体成像、病理切片等方法观察瘤体注射Cu S颗粒后经980nm近红外激光照射对肿瘤组织的杀灭、消融作用。利用ICP-OES观察小鼠胃肠道对亚微米级Cu S颗粒的吸收情况,并对小鼠胃肠道进行病理检测观察亚微米级Cu S颗粒对小鼠胃肠道的毒性作用。结果:1、980nm近红外激光以0.6W/cm2的功率照射四种不同粒径范围的Cu S颗粒溶液,溶液温度随着照射时间增加而增高、随着Cu S浓度的增加而增高,但粒径与温度变化没有明显的相关性。2、光热稳定性实验表明反复5次照射后所测Cu S溶液升温程度、升温速度一致,说明亚微米Cu S颗粒具有良好的光热稳定性。3、结肠癌细胞培养基中置入各样本Cu S,并经980nm近红外激光照射10min,测温仪所测得的温度同样也会随着Cu S液体浓度的增加而升高,但温度升高到一定程度后不会继续上升。4、CCK-8观察不同浓度Cu S溶液对结肠癌细胞生长情况影响时发现,浓度200ug/ml时,各样本组的细胞活性率均在80%以上;当浓度为400ug/ml时,四种粒径硫化铜对HCT116的生长开始产生影响,80-100nm Cu S组,100-200nm Cu S组,200-400nm Cu S组以及300-600nm Cu S组的细胞活性率依次为:70.2±1.3%,61.3±2.3%,57.9±2.01%,56.2±2.3%;当浓度达800ug/ml时,细胞活性下降至40%以下,80-100nm Cu S组,100-200nm Cu S组,200-400nm Cu S组以及300-600nm Cu S组的细胞活性率依次为:38.9±8.75%、25.7±2.8%,、26.8±5.6%、56.2±3.9%。5、利用Annexin V-FITC/PI检测近红外激光照射前后细胞凋亡情况发现,Cu S+HCT116+激光组与HCT116组、Cu S+HCT116组以及HCT116+激光组相比,表现出显著的肿瘤激光热杀伤效应(P0.05)。6、小动物活体荧光成像显示,瘤体注射Cu S颗粒后经980nm激光照射后移植瘤处荧光信号消失,证明对肿瘤有显著的杀灭消融效果,而空白对照组和蒸馏水瘤体注射对照组移植瘤处均可见荧光信号。7、ICP-OES检测Cu2+分布结果显示:粪液、胃组织、小肠组织和结肠组织中Cu2+含量分别占Cu S灌胃量的57%、3%、15%和8%,证明亚微米级Cu S颗粒通过灌胃方式进入胃肠道,大部分可通过粪便形式排泄出来。胃肠道组织病理检测证明未见明显的急性炎症毒性反应。结论:1、Cu S微粒具有良好的光热转化效率、光热稳定性,具有良好的光热治疗应用价值;2、经980nm近红外激光照射不同粒径、不同浓度硫化铜溶液后,其温度的变化与浓度和时间有关、与微粒粒径无关,升温均可达到42℃以上且温度升高速度平稳、变化幅度小,证明具有安全可靠的临床应用潜力。3、低浓度硫化铜微粒无显著细胞毒性,经近红外照射后产生光热效应可以显著杀灭肿瘤细胞,小动物活体实验证实对肿瘤组织有良好的消融效果,证明Cu S具有很好的光热治疗效果。4、亚微米级硫化铜胃肠道局部应用后大部分经粪便排出,胃肠道对亚微米级硫化铜颗粒具有很少的吸收作用且病理证实无显著毒性,说明亚微米级硫化铜颗粒消化道管腔局部喷洒应用这一给药途径具有较高的安全性。
[Abstract]:Objective: the digestive tract tumor is a global trend, threatening human health and causing huge social medical and economic burden. At present, early diagnosis and treatment is an effective way to prevent and control digestive tract tumors. Combining rapid development of nano probe and molecular imaging technology to realize integration of tumor diagnosis and treatment is an effective way to improve the detection rate of tumor and improve the effect of treatment. Method. In recent years, Photothermal therapy (PTT) has become a new model of integration of tumor diagnosis and treatment, showing great potential for transformation medicine and clinical application. Recent research shows that copper sulfide has high efficiency of photothermal transformation, and it has high signal to noise ratio of photoacoustic imaging contrast. It is a kind of photoacoustic imaging - light. In this study, by measuring the physicochemical properties of submicron copper sulfide in vitro, the photothermal effect of submicron copper sulphide was evaluated by 980nm near infrared laser irradiation of Cu S particles, and the effects of photothermal treatment on copper sulfide, biological toxicity and safety were analyzed by cell level and living animal level experiment. The clinical transformation and application of copper in the exploration of new directions and application patterns to explore new techniques for the integration of tumor diagnosis and treatment. Methods: using four submicron copper sulfide powders with different particle sizes to carry out experiments. The four sub micron grade Cu S particle sizes are about 80-100nm, 100-200nm, 200-400nm, and 300-600nm., respectively. The measurement of the properties of micron grade copper sulfide, in vitro cell experiments and in three parts of the small animal living experiment,.1, the particle size range of Cu S, the photothermal conversion properties of Cu S with different particle sizes, the influence of the concentration gradient and the laser irradiation time on the photothermal conversion temperature, and the determination of the photothermal stability of Cu S particles, and the observation of the photothermal transformation after multiple irradiation. The change of efficiency.2, the effect of Cu S submicron particles and colon cancer cells on the growth of living cells after incubation in vitro was observed by microscope. The cytotoxicity of four particle sizes and Cu S solutions with different concentrations of Cu S on colon cancer cells was observed by CCK-8 method. The cell culture medium containing Cu S particles with different concentrations were observed by 980nm near infrared laser and observed by 980nm near infrared laser. The relationship between the culture medium temperature and the concentration gradient of Cu S and the irradiation time. The apoptosis of the cells before and after the near infrared laser irradiation was analyzed by Annexin V-FITC/PI, and the killing effect of Cu S on the photothermal response was studied, and the subcutaneous xenograft of nude mice was prepared by the GFP-Luc SW480 colon cancer cell line, and the small animal body was carried out when the tumor grew to 7mm. Experiments were conducted to observe the killing and ablation of tumor tissues by intratumoral injection of Cu S particles by 980nm near infrared laser in vivo imaging of small animals and pathological sections. The absorption of submicron Cu S particles in the gastrointestinal tract of mice was observed by ICP-OES, and the submicron Cu S particles were observed by pathological examination of the gastrointestinal tract of mice. The toxic effect of the mouse gastrointestinal tract. Results: the 1980nm near infrared laser irradiated the Cu S particle solution of four different particle sizes with the power of 0.6W/cm2. The temperature of the solution increased with the increase of the irradiation time, and increased with the increase of Cu S concentration, but there was no obvious correlation.2 between the particle size and the temperature change, and the photothermal stability experiment showed repeated 5 times. The degree of temperature rising and heating rate of Cu S solution after irradiation are consistent, indicating that sub micron Cu S particles have good photothermal stability.3, Cu S is placed in the culture medium of colon cancer cells, and 10min by 980nm near infrared laser. The temperature measured by the thermometer will also increase with the increase of Cu S liquid concentration, but the temperature rises to the increase. A certain degree did not continue to increase.4, CCK-8 observed the effects of different concentrations of Cu S on the growth of colon cancer cells. When the concentration was 200ug/ml, the cell activity rate of each sample group was above 80%; when the concentration was 400ug/ml, four kinds of particle size copper sulfide had an influence on the growth of HCT116, 80-100nm Cu S group, 100-200nm Cu group, 200 The cell activity rates of -400nm Cu S group and 300-600nm Cu S group were 70.2 + 1.3%, 61.3 + 2.3%, 57.9 + 2.01%, 56.2 + 2.3%. When the concentration reached 800ug/ml, the cell activity decreased to less than 40%. The cell activity rate of 80-100nm Cu S group and 100-200nm Cu group was 38.9 + 8.75%, 25.7 + 2.8%, and 26.8 + .6%, 56.2 + 3.9%.5, using Annexin V-FITC/PI to detect the cell apoptosis before and after the near infrared laser irradiation, it was found that the Cu S+HCT116+ laser group showed significant tumor laser thermal killing effect (P0.05).6, compared with the HCT116 group, Cu S+HCT116 group and HCT116+ laser group. The fluorescence signal of the transplanted tumor disappeared after the laser irradiation. It was proved that the tumor had a significant effect on the ablation of the tumor. The fluorescence signal.7 was found in the blank control group and the injection control group of the distilled water tumor body. The ICP-OES detection Cu2+ distribution results showed that the content of Cu2+ in the fecal fluid, the gastric tissue, the small intestinal fabric and the colon tissue accounted for 57 of the Cu S gastric perfusion, respectively. %, 3%, 15% and 8% proved that submicron Cu S particles entered the gastrointestinal tract by gavage, most of which could be excreted in the form of feces. The pathological examination of the gastrointestinal tract showed no obvious acute inflammatory reaction. Conclusion: 1, Cu S particles have good photothermal conversion efficiency, photothermal stability, and good application price for photothermal treatment. 2, after irradiating different particle sizes and different concentrations of copper sulphide solution by 980nm near infrared laser, the temperature changes are related to the concentration and time, which are independent of the particle size, and the temperature can reach more than 42, and the temperature increases smoothly and the change range is small. It is proved that the potential of clinical application is safe and reliable, and the low concentration of copper sulfide particles is not significant. The effect of photothermal effect after near infrared irradiation can significantly kill tumor cells. In vivo experiments of small animals have proved to have good ablation effect on tumor tissue. It is proved that Cu S has a good effect of photothermal treatment.4. The submicron copper vulcanized gastric intestinal tract is discharged by the large part of the stomach and intestines, and the gastrointestinal tract is to submicron copper sulfide. The particles have little absorption and no significant toxicity is confirmed by pathology. It shows that the local spraying of the submicron copper sulfide particles in the digestive tract of the digestive tract has high safety.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735

【参考文献】