本文選題：納米技術(shù) + 光熱療法��； 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：目的：Cypate是具有光熱轉(zhuǎn)換效應(yīng)的有機(jī)材料,可應(yīng)用于腫瘤的光熱治療。本文構(gòu)建了一種新型有機(jī)光熱納米材料葉酸偶聯(lián)白蛋白Cypate(即FA-BSA-Cypate)納米顆粒,由于葉酸和cypate能同時結(jié)合于血清白蛋白的不同位點而被血清白蛋白運輸?shù)襟w內(nèi),因腫瘤的EPR效應(yīng)及葉酸與其受體的特異性結(jié)合而使膀胱腫瘤細(xì)胞更有效地攝取納米粒中cypate,體外及體內(nèi)實驗證實Folate-BSA-Cypate納米粒對膀胱腫瘤的靶向光熱殺傷效應(yīng),探究其作用機(jī)制,有望為后期臨床應(yīng)用提供理論基礎(chǔ)。方法：(1)合成有機(jī)材料Cypate并通過1H-NMR進(jìn)行表征,溶劑透析法制備葉酸偶聯(lián)白蛋白Cypate納米顆粒,透射電子顯微鏡(TEM)對葉酸偶聯(lián)白蛋白Cypate納米顆粒的形態(tài)進(jìn)行表征,動態(tài)光散射儀(DLS)測定納米粒粒徑分布,紫外分光光度法檢測納米粒載藥量和包封率,觀察不同濃度FA-BSA-Cypate納米粒溶液(0、1、10、100μg/ml)在808nm激光(1.5W/cm2)照射下溫度隨時間變化,證實FA-BSA-Cypate納米粒的光熱升溫效應(yīng)。(2)免疫熒光法檢測人膀胱癌5637細(xì)胞膜葉酸受體(FA-α receptor)的表達(dá),紫外分光光度法測定5637細(xì)胞對FA-BSA-Cypate納米粒、BSA-Cypate納米粒的攝取量隨時間變化,進(jìn)一步應(yīng)用激光共聚焦顯微鏡觀察5637細(xì)胞對FA-BSA-Cypate納米粒、BSA-Cypate納米粒的攝取情況。(3)MTT法比較不同濃度FA-BSA-Cypate納米粒細(xì)胞毒性,MTT法檢測不同F(xiàn)A-BSA-Cypate納米粒濃度、不同光照時間以及不同能量的808 nm的近紅光照射所誘導(dǎo)的對5637細(xì)胞的光熱殺傷效應(yīng)。(4)DHE(Dihydroethidium)染色考察FA-BSA-Cypate納米粒經(jīng)激光照射后在細(xì)胞內(nèi)單線態(tài)氧的水平；FA-BSA-Cypate納米粒聯(lián)合近紅外光照射情況下,檢測1,3-二苯基異苯并呋喃(DPBF)消耗量以反映的單線態(tài)氧(102)的生成。(5)設(shè)定為激光功率密度為1.5W/cm2、波長為808 nm的激光照射5 min；將細(xì)胞分為四組：空白對照組、激光組、BSA-Cypate+激光組、FA-BSA-Cypate+激光組；流式細(xì)胞術(shù)檢測各組細(xì)胞凋亡率；transwell法檢測細(xì)胞的遷移能力;qRT-PCR和Western-blot法檢測各組bcl-2、bax、 Caspase-3、Survivin、HSP70、HSP90mRNA和蛋白表達(dá)變化。(6)考察FA-BSA-Cypate納米粒在膀胱癌5637細(xì)胞荷瘤小鼠體內(nèi)的抗腫瘤活性,通過腫瘤體積增長、抑瘤率對光熱抗腫瘤效果進(jìn)行評價。結(jié)果：成功制備了具有良好光熱升溫特性的FA-BSA-Cypate納米粒,其粒徑為(101.2±9.3)nm、載藥量為(45.8±3.3)%、包封率(91.5±4.2)%；808nm激光(1.5W/cm2,5min)照射下,納米粒溶液最高溫度可達(dá)59℃,且具有濃度依賴性；細(xì)胞免疫熒光證實人膀胱癌5637細(xì)胞膜表達(dá)葉酸受體(FA-a receptor);細(xì)胞攝取試驗表明FA-BSA-Cypate納米粒對膀胱癌5637細(xì)胞的靶向性；MTT顯示不同濃度的FA-BSA-Cypate納米粒作用于5637細(xì)胞24h后細(xì)胞存活率在95%以上,無明顯生物毒性：聯(lián)合808nm激光照射下,細(xì)胞存活率隨著納米粒濃度和激光能量密度的增加而下降,細(xì)胞存活率也隨著激光照射時間的延長而下降；近紅外光照條件下,FA-BSA-Cypate納米粒能夠促進(jìn)單線態(tài)氧的生成,加重了細(xì)胞毒性；流式細(xì)胞檢測結(jié)果顯示FA-BSA-Cypate納米粒組細(xì)胞凋亡率顯著增高；transwell法檢測結(jié)果表明FA-BSA-Cypate納米粒組細(xì)胞的遷移能力顯著下降；qRT-PCR及Western blot結(jié)果顯不,FA-BSA-Cypate+激光組Bax、Caspase-3、Hsp70、Hsp90的表達(dá)顯著增高,而BcL-2、Survivin的表達(dá)明顯降低。808 nm激光照射(1.5 W/cm2)下,FA-BSA-Cypate納米粒對5637細(xì)胞荷瘤裸鼠的皮下腫瘤生長具有顯著的抑制作用。結(jié)論：有機(jī)納米材料FA-BSA-Cypate納米粒毒性低、腫瘤細(xì)胞靶向性強(qiáng)；體外及體內(nèi)實驗表明FA-BSA-Cypate納米粒介導(dǎo)的靶向光熱療法對膀胱癌具有良好的靶向殺傷效果,能有效抑制5637細(xì)胞的增殖并促進(jìn)其凋亡。本研究為將來臨床應(yīng)用基于FA-BSA-Cypate納米粒的膀胱癌的靶向光熱治療奠定了一定的理論基礎(chǔ)。
[Abstract]:Objective : Cypate was an organic material with photo - heat transfer effect . It was used in the photothermal treatment of tumor . The effect of folate - BSA - Cypate nanoparticles on the tumor of bladder cancer was measured by UV spectrophotometry . The effect of Folate - BSA - Cypate nanoparticles on bladder tumor was investigated . The effects of different concentrations of FA - BSA - Cypate nanoparticles were observed .
In the case of near - infrared irradiation , FA - BSA - Cypate nanoparticles were used to detect the consumption of 1,3 - diphenylisobenzofuran ( DPBF ) to reflect the generation of singlet oxygen ( 102 ) .
The cells were divided into four groups : blank control group , laser group , BSA - Cypate + laser group , FA - BSA - Cypate + laser group ;
Flow cytometry was used to detect the apoptosis rate of each group .
The expression of bcl - 2 , bax , Caspase - 3 , Survivin , HSP70 , HSP90mRNA and protein were detected by transwell method .
Under the irradiation of 808 nm laser ( 1.5W / cm ~ 2 , 5 min ) , the highest temperature of the nanoparticle solution can reach 59 鈩，

本文編號：2111840