【作者】 陈怡；

【导师】 张宇；

【作者基本信息】 东南大学 ， 生物医学工程， 2020， 硕士

【摘要】 急性髓系白血病(AML)的耐药和复发是目前AML的治疗瓶颈,因此解决AML耐药性的问题对于降低其死亡率具有重大意义,其中的关键技术问题之一是AML耐药细胞的捕获与检测。目前主流细胞分选方法主要是流式细胞分选和免疫磁珠细胞分选。然而流式细胞分选仪器昂贵、操作技术难度高,通常需要超过105个细胞作为起始上样量;免疫磁珠细胞分选通常需要配合高梯度磁性细胞分离柱使用,且其达到的强磁场可能会吸引本身有磁性的细胞聚集在分离柱中,导致细胞的非特异性捕获。针对目前急性髓系白血病的耐药与复发现状以及当前细胞分选方法存在的局限,本文设计了一种高效的基于磁性纳米探针的微流控细胞分选系统(Magnetic nanoparticle based microfluidic cell sorting,MMCS),将磁性纳米探针与微流控芯片结合,可在简单的永磁体下实现AML耐药细胞的捕获与检测,突破了现有细胞分选方法的局限性,为发展新型AML耐药治疗策略与检测技术奠定基础。首先采用高温热解法成功制备10、15、20 nm的油溶性Fe3O4纳米颗粒,并通过改进制备工艺将Fe3O4纳米颗粒合成产量提升至1 g,产率大于89%。通过旋转蒸发法将DSPE-PEG修饰在纳米颗粒表面,得到水溶性Fe3O4@PEG纳米颗粒,其具有较高的饱和磁化强度、良好的稳定性及批间一致性。选择饱和磁化强度最高(93 emu/g[Fe])且稳定性好的20 nm Fe3O4纳米颗粒进行后续的细胞磁分选。然后构建了两种不同的探针,对两种纳米探针的细胞捕获能力进行探索与研究,选出高性能的纳米探针。一是基于多肽的纳米探针,利用链霉亲和素-生物素放大体系,将链霉亲和素、荧光分子Cy5、多肽E5组装在氧化铁纳米颗粒表面,最大限度增加了荧光分子和多肽的负载量。细胞普鲁士蓝染色结果表明未修饰荧光分子的Fe3O4@PEG@SA@E5纳米探针对CXCR4的特异性靶向能力优异,而修饰荧光分子的Fe3O4@PEG@SA-Cy5@E5纳米探针靶向HL-60细胞的能力显著降低。通过实验对其原因进行探讨,发现Cy5能与E5共价结合,使得Cy5荧光淬灭,多肽空间结构改变,造成多肽靶向能力大幅减弱。因此制备了荧光分子负载的二氧化硅包裹的氧化铁纳米颗粒Fe3O4@SiO2/Cy5,将Cy5偶联在二氧化硅层中间,其与E5混合后荧光强度不变,验证了利用此纳米颗粒构建纳米探针实现细胞捕获与荧光检测的可行性。二是基于抗体的纳米探针,分别将单克隆抗体12G5、荧光分子F647修饰在氧化铁纳米颗粒表面,构建用于细胞捕获的纳米探针(Fe3O4@PEG@12G5-F647),该纳米探针被证明具有优异的特异性细胞靶向能力。确定了最优的细胞分选条件为:5*105个HL-60细胞中加入40μg高价态Fe3O4@PEG@12G5-F647纳米探针(表面偶联12个抗体),37℃孵育1 h。Fe3O4@PEG@12G5-F647纳米探针的细胞捕获效率较高(96%~100%),细胞分选纯度高达98%,且实验证明靶细胞的检测与阴性细胞的数量无关,说明该方法具有较强的抗干扰能力。比较这两种纳米探针的细胞捕获能力,其中基于抗体的纳米探针Fe3O4@PEG@12G5-F647对于CXCR4的特异性靶向能力更强,因此选择高性能的基于抗体的纳米探针与微流控芯片联合,实现AML耐药细胞的捕获与检测。最后将高性能纳米探针与微流控芯片结合,构建基于磁性纳米探针的微流控细胞分选系统(MMCS),可在简单的永磁体下实现AML耐药细胞的捕获,采用荧光定量分析仪实现耐药细胞数量的定量检测。设计了共两个版本的微流控芯片,其中优化后的第二版微流控芯片能够更有效地截留磁标记的靶细胞,其细胞捕获率与第一版微流控芯片相比显著提高。确定最优的细胞流速为10μL/min,细胞捕获率为90.4%。微流控细胞检测的线性范围为104~5*105个细胞(R2为0.990),建立了微流控细胞捕获与检测的标准曲线。确定微流控细胞检测的检测限为1.53*103个。在模拟样本的细胞捕获与检测中,检测结果与真实细胞数量之间具有很好的一致性(R2大于0.990),且两者之间的相对偏差小于10%,证明了微流控耐药细胞捕获与检测方法具有较高的准确度。总之,成功开发了用于AML耐药细胞捕获和检测的基于磁性纳米探针的微流控细胞分选系统(MMCS),克服了当前细胞分选方法的局限,具有成本低、操作简单、不需要强梯度磁场、细胞分选效率与分选纯度高、仪器可便携、分选过程自动化的优势,具有便携性和临床应用的潜力。更多还原

【Abstract】 The drug resistance and recurrence is the bottleneck in the treatment of acute myeloid leukemia(AML).One of the key technologies to solve the problem is the capture and detection of drug-resistant cells.The current mainstream cell sorting methods are flow cytometry cell sorting and immunomagnetic bead cell sorting.However,flow cytometry instruments are expensive and difficult to operate,and usually require more than 105 cells as the initial sample load.Immunemagnetic bead cell sorting usually requires high gradient magnetic field,which can reach strong magnetic field and may result in non-specific capture of cells with magnetic properties inherently.Here,we report a highly efficient magnetic nanoprobe based microfluidic cell sorting system(MMCS).MMCS system combines magnetic nanoprobe and microfluidic for the capture and detection of drug-resistant cells under simple permanent magnets,breaking the limitations of existing cell sorting methods.Firstly,multiple sizes of Fe3O4@OA nanoparticles(10,15,20 nm)were successfully prepared by thermal decomposition method.By improving the preparation process,the synthesis yield of Fe3O4 nanoparticles was increased to 1 g,with a yield greater than 89%.Water-soluble Fe3O4@PEG nanoparticles were obtained by the modification of amphiphilic DSPE-PEG,which has high saturation magnetization,good stability and superior batch-tobatch consistency.By comparision,20 nm Fe3O4 nanoparticle which was stable and had the highest saturation magnetization(93 emu/g [Fe])was selected for subsequent cell sorting.Secondly,two kinds of different nanoprobes were constructed and their cell capture ability were studied to select a high-performance nanoprobe.One was peptide-based nanoprobe.Streptavidin-biotin amplification system was used to assemble streptavidin and peptide on the surface of Fe3O4 nanoparticles in the construction of peptide-based nanoprobe,which maximized the loading of fluorescent molecules and peptides.Prussian blue staining results showed that the unmodified fluorescent molecule Fe3O4@PEG@SA@E5 nanoprobe had excellent specific targeting ability to CXCR4,while the cell targeting ability of the modified fluorescent molecule Fe3O4@PEG@SA-Cy5@E5nanoprobe was significantly reduced.It was found that Cy5 can covalently bind to E5,which can quench the fluorescence of Cy5 and change the spatial structure of the peptide,resulting in a significant reduction of the targeting ability of peptide E5.Therefore,the silica-coated iron oxide nanoparticles Fe3O4@SiO2/Cy5 were prepared,in which Cy5 was coupled in the middle of the silica layer.After mixing with E5,the fluorescence intensity of the mixture remained unchanged,which verified the feasibility of constructing nanoprobe to achieve cell capture and detection.The other was an antibody-based nanoprobe.Monoclonal antibody 12G5 and fluorescent molecule F647 was modified on the surface of iron oxide nanoparticles successively to construct Fe3O4@PEG@12G5-F647 nanoprobe,which was proved to have excellent specific cell targeting ability.The optimal cell sorting conditions were determined as follows: 40 μg of Fe3O4@PEG@12G5-F647 nanoprobe(12 antibodies conjugated)was added to 5*105 HL-60 cells and incubated at 37°C for 1 h.The Fe3O4@PEG@12G5-F647 nanoprobe had a high cell capture efficiency(96% ~ 100%),and the cell sorting purity was as high as 98%.Besides,the capture and detection of target cells has nothing to do with the number of negative cells,indicating that this method has strong anti-interference ability.Among the two kinds of nanoprobes,antibody-based nanoprobe was demonstrated to have distinguished cell-targeting ability with a cell capture efficiency of more than 96%.Therefore,antibody-based nanoprobe was selected to combine with the microfluidic chip,achieving the capture and detection of AML drug-resistant cells.Finally,the combination of magnetic nanoprobe and microfluidic(MMCS)was used to capture drug-resistant cells under a permanent magnet,and the number of drug-resistant cells was detected by a fluorescence quantitative analyzer.A total of two versions of microfluidic chip were designed.The optimized version of microfluidic chip can capture target cells more effectively,with a higher cell capture effiency compared with the original version of microfluidic chip.The optimal cell flow rate was determined to be 10 μL/min,and the cell capture rate reached to 90.4%.The linear range of microfluidic cell detection was 104~5*105cells(R2 was 0.990).A standard curve for microfluidic cell capture and detection was also established.The detection limit was determined to be 1.53*103.In the cell capture and detection of simulated samples,the detected cell number was consistent with the actual cell number of HL-60(R2 was greater than 0.990),and the relative deviation is less than 10%,proving that the detection of drug-resistant cells by microfluidic had a high accuracy.In conclusion,MMCS system for the capture and detection of drug-resistant cell in acute myeloid leukemia has been successfully developed,which overcomes the limitations of current cell sorting methods.MMCS system has the advantages of low cost,simple operation,no strong gradient magnetic field,high cell sorting efficiency,high cell sorting purity,and automated sorting process,which is portable and has potential for widespread clinical application.更多还原