靶向胶质瘤EGFRvⅢ核酸适配体—量子点成像体内外研究

发布时间：2018-04-10 12:44

本文选题：量子点 + 适配子　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:探究量子点(QDs)连接适配子(QD-Apt)靶向EGFRv III胶质瘤成像的方法与可行性。方法:1.QD-Apt的合成、物理学特征测定及安全性研究1)QD-Apt的合成及物理学特征测定:生物素修饰的适配子(A32)与链霉亲和素修饰的量子点(SA-QDs)通过生物素-链霉亲和素链接将两者结合形成QD-Apt。Zetasizer Nano ZS型纳米粒度分析仪测定QD-Apt粒径,荧光分光光度计测定QD-Apt及QDs的吸收光谱和发射光谱。琼脂糖凝胶电泳初步验证适配子与量子点的连接。2)安全性研究:体外采用CCK-8法检测QD-Apt对HUVEC、U87和U87-EGFRv III细胞活力的影响。体内尾静脉注射QD-Apt后,不同时间点测量裸鼠体重。HE染色观察裸鼠各器官组织的病理变化。2.QD-Apt在体外的靶向荧光标记1)EGFRv III在细胞株中的表达:逆转录PCR(RT-PCR)、western blot和细胞免疫荧光方法检测HUVEC、U87、U87-EGFRv III细胞ERFRv III的表达。2)QD-Apt靶向胶质瘤细胞株的研究:QD-Apt标记HUVEC、U87、U87-EGFRv III细胞,并在激光共聚焦下观察荧光强度。流式细胞术检测QD-Apt对HUVEC、U87、U87-EGFRv III细胞的标记率。3.QD-Apt在裸鼠体内成像及组织分布:建立裸鼠颅内胶质瘤模型并用MRI检测肿瘤生长情况。4周后,尾静脉注射QD-Apt及QDs,6小时后进行活体成像。并取瘤组织及正常脑组织行冰冻切片并在激光共聚焦下观察;分光光度计测定各组织器官的荧光强度。结果:1.成功构建QD-Apt纳米探针,QD-Apt及QDs的粒径均为20nm左右,QDs与A32连接后,其最大发射光谱仍为605±5nm,吸收光谱波峰仍为330nm。琼脂糖凝胶电泳结果表明,适配子与量子点连接成功。体内外的安全性研究均证实QD-Apt无明显毒性。2.EGFRv III在U87-EGFRv III细胞中为阳性表达,而在U87、HUVEC中均为阴性表达。QD-Apt可特异性的结合U87-EGFRv III胶质瘤细胞株并且标记率高达81.53%。3.活体荧光成像显示,QD-Apt可以透过血脑屏障并通过特异性结合EGFRv III而选择性地聚集在肿瘤组织中。与正常脑组织相比,肿瘤组织中具有很强的荧光信号,可以清晰地显示出肿瘤的边界。QD-Apt主要聚集在EGFRv III(+)的肿瘤组织、肝脏和肾脏,而在瘤旁、正常脑组织、脾、肺和心等较少。结论:QD-Apt可以通过特异性结合EGFRv III靶向胶质瘤荧光成像。
[Abstract]:Aim: to investigate the feasibility and method of QD-Apt targeting EGFRv III glioma imaging.Method: 1. Synthesis of QD-Apt,Synthesis and physical characterization of 1)QD-Apt: biotin modified aptamer A32) and Streptomycin modified Quantum Dot SA-QDs) combine the two to form QD-Apt.Zetasizer via biotin-streptavidin linkageNano ZS nano-particle size analyzer was used to measure the particle size of QD-Apt.The absorption and emission spectra of QD-Apt and QDs were measured by fluorescence spectrophotometer.Agarose gel electrophoresis (agarose gel electrophoresis) was used to preliminarily verify the safety of aptamer and quantum dots (QDs). The effects of QD-Apt on the viability of HUVEC-U87 and U87-EGFRv III cells were detected by CCK-8 assay in vitro.After intravenous injection of QD-Apt in vivo,Study on the expression of ERFRv III. QD-Apt targeted glioma cell line; U87-EGFRv III cell line U87-EGFRv, U87and U87-EGFRv III cells labeled with: QD-Apt.Fluorescence intensity was observed in confocal laser.Frozen sections of tumor tissue and normal brain tissue were taken and observed under confocal laser. Fluorescence intensity of various tissues and organs were measured by spectrophotometer.The result is 1: 1.After successfully constructing QD-Apt nanoprobe QD-Apt and QDs, the maximum emission spectrum and absorption peak of QD-Apt and QDs were 605 卤5nmand 330nmrespectively after the QDs and A32 were connected with 20nm or so.Agarose gel electrophoresis showed that the aptamer was successfully connected with quantum dots.In vivo and in vitro safety studies confirmed that QD-Apt was not virulent. 2. EGFRv III was positive in U87-EGFRv III cells, but negative expression in U87 HUVEC. QD-Apt could specifically bind U87-EGFRv III glioma cells and the labeling rate was as high as 81.53. 3.In vivo fluorescence imaging showed that QD-Apt could selectively gather in tumor tissues through the blood-brain barrier and by specifically binding to EGFRv III.Compared with normal brain tissue, tumor tissue has strong fluorescence signal, which clearly shows that the boundary of tumor. QD-Apt is mainly clustered in tumor tissue, liver and kidney of EGFRv III (), while in adjacent tumor, normal brain tissue, spleen,The lungs and the heart are less.Conclusion: QD-Apt can target glioma with specific binding to EGFRv III.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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