本文選題：硫化銅 + 光熱治療　； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:消化道腫瘤呈全球高發(fā)趨勢(shì),威脅人類(lèi)的健康,造成巨大的社會(huì)醫(yī)療經(jīng)濟(jì)負(fù)擔(dān)。目前,早診早治是防治消化道腫瘤的有效方式,結(jié)合快速發(fā)展納米探針與分子影像技術(shù)實(shí)現(xiàn)腫瘤診療一體化,是提高腫瘤檢出率、提高治療效果的有效方法。近年來(lái),光熱治療(Photothermal therapy,PTT)已成為一種腫瘤診療一體化新模式,展現(xiàn)了巨大的轉(zhuǎn)化醫(yī)學(xué)和臨床應(yīng)用潛力。近期研究表明硫化銅具有高效的光熱轉(zhuǎn)化效能,同時(shí)具有高信噪比的光聲成像對(duì)比度,是一種可以實(shí)現(xiàn)光聲成像-光熱治療的診療一體化介質(zhì)。本研究通過(guò)離體測(cè)定亞微米級(jí)硫化銅理化性質(zhì)、980nm近紅外激光照射Cu S顆粒光熱轉(zhuǎn)化效率評(píng)估亞微米級(jí)硫化銅光熱效應(yīng),通過(guò)細(xì)胞水平和活體動(dòng)物水平實(shí)驗(yàn)分析硫化銅光熱治療效果、生物毒性和安全性,為硫化銅的臨床轉(zhuǎn)化應(yīng)用探尋新的方向與應(yīng)用模式,為腫瘤診療一體化探索新的技術(shù)。方法:利用四種不同粒徑范圍的亞微米級(jí)硫化銅粉末進(jìn)行實(shí)驗(yàn)探究。這四種亞微米級(jí)Cu S粒徑范圍分別約為80-100nm,100-200nm,200-400nm,300-600nm。實(shí)驗(yàn)內(nèi)容包括亞微米級(jí)硫化銅的性質(zhì)測(cè)定、體外細(xì)胞實(shí)驗(yàn)以及小動(dòng)物活體實(shí)驗(yàn)三個(gè)部分。1、電鏡觀察Cu S粒徑范圍,測(cè)定不同粒徑Cu S光熱轉(zhuǎn)換性質(zhì),觀察濃度梯度以及激光照射時(shí)間對(duì)光熱轉(zhuǎn)換溫度的影響;測(cè)定Cu S微粒光熱穩(wěn)定性,觀察多次照射后光熱轉(zhuǎn)換效率變化情況。2、顯微鏡體外觀察將Cu S亞微米顆粒與結(jié)腸癌細(xì)胞孵育后對(duì)活細(xì)胞生長(zhǎng)的影響,利用CCK-8法觀察四種粒徑、不同濃度的Cu S溶液對(duì)結(jié)腸癌細(xì)胞的細(xì)胞毒性。用980nm近紅外激光照射含不同濃度Cu S顆粒的細(xì)胞培養(yǎng)基10min,觀察培養(yǎng)基溫度與Cu S濃度梯度、照射時(shí)間的關(guān)系。利用Annexin V-FITC/PI對(duì)近紅外激光照射前后的細(xì)胞凋亡情況進(jìn)行分析,研究Cu S光熱效應(yīng)對(duì)的殺傷作用。3、利用GFP-Luc SW480結(jié)腸癌細(xì)胞株制備裸鼠皮下移植瘤,腫瘤長(zhǎng)至7mm左右時(shí)進(jìn)行小動(dòng)物活體實(shí)驗(yàn),通過(guò)小動(dòng)物活體成像、病理切片等方法觀察瘤體注射Cu S顆粒后經(jīng)980nm近紅外激光照射對(duì)腫瘤組織的殺滅、消融作用。利用ICP-OES觀察小鼠胃腸道對(duì)亞微米級(jí)Cu S顆粒的吸收情況,并對(duì)小鼠胃腸道進(jìn)行病理檢測(cè)觀察亞微米級(jí)Cu S顆粒對(duì)小鼠胃腸道的毒性作用。結(jié)果:1、980nm近紅外激光以0.6W/cm2的功率照射四種不同粒徑范圍的Cu S顆粒溶液,溶液溫度隨著照射時(shí)間增加而增高、隨著Cu S濃度的增加而增高,但粒徑與溫度變化沒(méi)有明顯的相關(guān)性。2、光熱穩(wěn)定性實(shí)驗(yàn)表明反復(fù)5次照射后所測(cè)Cu S溶液升溫程度、升溫速度一致,說(shuō)明亞微米Cu S顆粒具有良好的光熱穩(wěn)定性。3、結(jié)腸癌細(xì)胞培養(yǎng)基中置入各樣本Cu S,并經(jīng)980nm近紅外激光照射10min,測(cè)溫儀所測(cè)得的溫度同樣也會(huì)隨著Cu S液體濃度的增加而升高,但溫度升高到一定程度后不會(huì)繼續(xù)上升。4、CCK-8觀察不同濃度Cu S溶液對(duì)結(jié)腸癌細(xì)胞生長(zhǎng)情況影響時(shí)發(fā)現(xiàn),濃度200ug/ml時(shí),各樣本組的細(xì)胞活性率均在80%以上;當(dāng)濃度為400ug/ml時(shí),四種粒徑硫化銅對(duì)HCT116的生長(zhǎng)開(kāi)始產(chǎn)生影響,80-100nm Cu S組,100-200nm Cu S組,200-400nm Cu S組以及300-600nm Cu S組的細(xì)胞活性率依次為:70.2±1.3%,61.3±2.3%,57.9±2.01%,56.2±2.3%;當(dāng)濃度達(dá)800ug/ml時(shí),細(xì)胞活性下降至40%以下,80-100nm Cu S組,100-200nm Cu S組,200-400nm Cu S組以及300-600nm Cu S組的細(xì)胞活性率依次為:38.9±8.75%、25.7±2.8%,、26.8±5.6%、56.2±3.9%。5、利用Annexin V-FITC/PI檢測(cè)近紅外激光照射前后細(xì)胞凋亡情況發(fā)現(xiàn),Cu S+HCT116+激光組與HCT116組、Cu S+HCT116組以及HCT116+激光組相比,表現(xiàn)出顯著的腫瘤激光熱殺傷效應(yīng)(P0.05)。6、小動(dòng)物活體熒光成像顯示,瘤體注射Cu S顆粒后經(jīng)980nm激光照射后移植瘤處熒光信號(hào)消失,證明對(duì)腫瘤有顯著的殺滅消融效果,而空白對(duì)照組和蒸餾水瘤體注射對(duì)照組移植瘤處均可見(jiàn)熒光信號(hào)。7、ICP-OES檢測(cè)Cu2+分布結(jié)果顯示:糞液、胃組織、小腸組織和結(jié)腸組織中Cu2+含量分別占Cu S灌胃量的57%、3%、15%和8%,證明亞微米級(jí)Cu S顆粒通過(guò)灌胃方式進(jìn)入胃腸道,大部分可通過(guò)糞便形式排泄出來(lái)。胃腸道組織病理檢測(cè)證明未見(jiàn)明顯的急性炎癥毒性反應(yīng)。結(jié)論:1、Cu S微粒具有良好的光熱轉(zhuǎn)化效率、光熱穩(wěn)定性,具有良好的光熱治療應(yīng)用價(jià)值;2、經(jīng)980nm近紅外激光照射不同粒徑、不同濃度硫化銅溶液后,其溫度的變化與濃度和時(shí)間有關(guān)、與微粒粒徑無(wú)關(guān),升溫均可達(dá)到42℃以上且溫度升高速度平穩(wěn)、變化幅度小,證明具有安全可靠的臨床應(yīng)用潛力。3、低濃度硫化銅微粒無(wú)顯著細(xì)胞毒性,經(jīng)近紅外照射后產(chǎn)生光熱效應(yīng)可以顯著殺滅腫瘤細(xì)胞,小動(dòng)物活體實(shí)驗(yàn)證實(shí)對(duì)腫瘤組織有良好的消融效果,證明Cu S具有很好的光熱治療效果。4、亞微米級(jí)硫化銅胃腸道局部應(yīng)用后大部分經(jīng)糞便排出,胃腸道對(duì)亞微米級(jí)硫化銅顆粒具有很少的吸收作用且病理證實(shí)無(wú)顯著毒性,說(shuō)明亞微米級(jí)硫化銅顆粒消化道管腔局部噴灑應(yīng)用這一給藥途徑具有較高的安全性。
[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.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735

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