结果1.检测流程本研究将对入组的患有结直肠癌的患者进行外周血血液样本及

结果1.检测流程本研究将对入组的患有结直肠癌的患者进行外周血血液样本及与之对应的组织样本进行检测，所选用的IML制备流程如图1所示，抗体与GHDC反应形成抗体-GHDC化合物衍生物，再结合DOPC、胆固醇和Fe3O4-HMN采用反相一步法制备MIL。在获得了循环肿瘤细胞来源的DNA以及组织来源的DNA之后所进行的后续检测方案亦如图1所示。通过与公共数据库中的数据进行比对，获悉目标miRNA在结直肠癌中呈现下调表达，设计一系列实验进行验证，并对目标miRNA在结直肠癌发生发展过程的相关作用进行研究。CTC分离检测系统的性能评价对于IMLs的微观结构和物理化学表征如图2所示。MIL的紫外吸收光谱如图2A所示，EpCAM-IML和EGFR-IML在280 nm附近有清晰的紫外吸收峰。紫外吸收结果证明磁性纳米颗粒表面已建立抗体，纳米颗粒表面抗体含量为0.1mg/mg。可从磁饱和曲线（图2B）看出制备的微球具有较高的饱和磁化强度，显示出超顺磁特性，可以有效地用于肿瘤细胞的分离。使用粒度分析仪检测粒径的变化，结果表明EpCAM抗体和EGFR抗体修改之后分别为180 nm 和202 nm (图2C)。粒径小，有助于提高抗体磁性脂质体在水溶液中的稳定性，本研究制备的EpCAM 和EGFR脂质磁珠符合预期要求。此外，EpCAM -IML和EGFR-IML的表面电位为0.06±0.02 mV，为中性，可以减少前人研究报道的正电流引起的非特异性吸附的干扰 (图2D)。在原子力显微镜下，两种粒子具有相似的球形，但分布均匀性相对较低，颗粒直径大于100 nm，颗粒表面较粗（图2E-2F）。 IML对结直肠癌细胞的靶向识别能力对本实验制备的EpCAM-MIL细胞毒性分析显示，在相同浓度的EpCAM MIL下，随着处理时间的延长，SW480和LOVO细胞的存活率逐渐下降。当EpCAM-MIL的浓度高于200μg/mL时，其对结直肠癌细胞的细胞毒性逐渐升高（图3A-3B）。以上结果表明两种IML对肿瘤细胞具有相同的抑制作用，抑制能力仅与添加IML的浓度有关。激光扫描共聚焦显微镜结果显示，SW480细胞细胞核内的红色荧光强度明显高于LOVO细胞，说明APC在SW480细胞中的表达水平明显低于PC-3细胞(图3C)。从结直肠癌患者血样中分离CTC，荧光抗体染色后用荧光显微镜观察。成像结果如图3D所示，细胞在白光下与MIL结合，细胞周围有明显的磁性纳米颗粒。CK8、18、19-FITC呈绿色高阳性荧光，DAPI呈蓝色高阳性荧光，CD45呈阴性，直径大于8 μm的细胞可判定为CTC。当肿瘤大小为50 mm3、100 mm3、200mm3时，外周血中CTC计数分别为4 /mL、19 /mL、37 /mL（图3E）。

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结果 (英语) 1: [复制]

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Results 1. Detection process This study will test the peripheral blood blood samples and corresponding tissue samples of the enrolled patients with colorectal cancer. The selected IML preparation process is shown in Figure 1. The antibody reacts with GHDC The antibody-GHDC compound derivative is formed, and then combined with DOPC, cholesterol and Fe3O4-HMN, MIL is prepared by a reverse phase one-step method. The follow-up detection scheme after obtaining DNA from circulating tumor cells and DNA from tissue is also shown in Figure 1. By comparing with the data in public databases, it is learned that the target miRNA is down-regulated in colorectal cancer, a series of experiments are designed to verify, and the relevant role of the target miRNA in the occurrence and development of colorectal cancer is studied. The performance evaluation of CTC separation detection system for the microstructure and physical and chemical characterization of IMLs is shown in Figure 2. The UV absorption spectrum of MIL is shown in Figure 2A. EpCAM-IML and EGFR-IML have clear UV absorption peaks near 280 nm. The UV absorption results prove that antibodies have been established on the surface of the magnetic nanoparticles, and the antibody content on the surface of the nanoparticles is 0.1 mg/mg. It can be seen from the magnetic saturation curve (Figure 2B) that the prepared microspheres have high saturation magnetization, show superparamagnetic properties, and can be effectively used for tumor cell separation. A particle size analyzer was used to detect the change in particle size, and the results showed that EpCAM antibody and EGFR antibody were modified to be 180 nm and 202 nm, respectively (Figure 2C). The small particle size helps to improve the stability of antibody magnetic liposomes in aqueous solution. The EpCAM and EGFR lipid magnetic beads prepared in this study meet the expected requirements. In addition, the surface potential of EpCAM-IML and EGFR-IML is 0.06±0.02 mV, which is neutral, which can reduce the interference of non-specific adsorption caused by positive current reported in previous studies (Figure 2D). Under the atomic force microscope, the two kinds of particles have similar spherical shapes, but the distribution uniformity is relatively low, the particle diameter is greater than 100 nm, and the particle surface is relatively coarse (Figure 2E-2F). The cytotoxicity analysis of EpCAM-MIL prepared by IML for colorectal cancer cells showed that under the same concentration of EpCAM MIL, the survival rate of SW480 and LOVO cells gradually decreased with the extension of treatment time. When the concentration of EpCAM-MIL is higher than 200μg/mL, its cytotoxicity to colorectal cancer cells gradually increases (Figure 3A-3B). The above results indicate that the two kinds of IML have the same inhibitory effect on tumor cells, and the inhibitory ability is only related to the concentration of IML added. The results of laser scanning confocal microscopy showed that the red fluorescence intensity in the nucleus of SW480 cells was significantly higher than that in LOVO cells, indicating that the expression level of APC in SW480 cells was significantly lower than that in PC-3 cells (Figure 3C). CTC was separated from blood samples of patients with colorectal cancer, stained with fluorescent antibodies, and observed with a fluorescent microscope. The imaging results are shown in Figure 3D. The cells bind to MIL under white light, and there are obvious magnetic nanoparticles around the cells. CK8, 18, 19-FITC showed green high-positive fluorescence, DAPI showed blue high-positive fluorescence, CD45 was negative, and cells with a diameter greater than 8 μm could be judged as CTC. When the tumor size was 50 mm3, 100 mm3, and 200 mm3, the CTC counts in the peripheral blood were 4 /mL, 19 /mL, and 37 /mL (Figure 3E).

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结果 (英语) 2:[复制]

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Results. 1. Detection process In this study, patients with colorectal cancer in the group will be tested for exosorcular blood samples and corresponding tissue samples, the selected IML preparation process is shown in Figure 1, antibodies and GHDC reactions to form antibody-GHDC compound derivatives, and then combined with DOPC, cholesterol and Fe3O4-HMN using inverse one-step legal preparation MIL. Subsequent tests were also shown in Figure 1 after DNA from circulating tumor cell sources and DNA from tissue sources was obtained. By comparing with the data in the public database, it is learned that the target miRNA presents downward expression in colorectal cancer, designs a series of experiments to verify, and studies the role of target miRNA in the development of colorectal cancer. Performance evaluation of CTC separation detection system The microstructure and physical and chemical characteristics of IMLs are shown in Figure 2. The UV absorption spectrum of MIL is shown in Figure 2A, and EpCAM-IML and EGFR-IML have clear UV absorption peaks around 280 nm. Ultraviolet absorption results show that antibodies have been established on the surface of magnetic nanoparticles, with an antibody content of 0.1 mg/mg on the surface of nanoparticles. It can be seen from the magnetic saturation curve (Figure 2B) that the prepared microcircule has a high saturation magnetization strength, showing super-flugic properties, which can be used effectively for the separation of tumor cells. The particle size analyzer was used to detect particle size changes, and the results showed that the EpCAM antibody and the EGFR antibody were modified to 180 nm and 202 nm, respectively (Figure 2C). The small particle size helps to improve the stability of antibody magnetic lipids in aqueous solutions, and the EpCAM and EGFR lipid beads prepared in this study meet the expected requirements. In addition, epCAM-IML and EGFR-IML have surface capacities of 0.06±0.02 mV, which is neutral and reduces nonse specific adsorption interference caused by positive currents reported in previous studies (Figure 2D). Under the atomic force microscope, the two particles have similar spherical shapes, but have relatively low distribution uniformity, particle diameters greater than 100 nm, and thicker particle surfaces (Figure 2E-2F). IML's ability to target colorectal cancer cells The analysis of EpCAM-MIL cytotoxicity prepared for this experiment showed that the survival rate of SW480 and LOVE cells decreased gradually with the extension of treatment time at epCAM MIL at the same concentration. When the concentration of EpCAM-MIL is higher than 200 μg/mL, its cytotoxicity to colorectal cancer cells gradually increases (Figure 3A-3B). The above results show that the two IMLs have the same inhibitory effect on tumor cells, and the inhibition ability is only related to the concentration of added IML. Laser scanning concentred microscope results showed that the red fluorescence intensity in the nucleus of SW480 cells was significantly higher than that of LOVE cells, indicating that the expression level of APC in SW480 cells was significantly lower than that of PC-3 cells (Figure 3C). CTC was isolated from blood samples of colorectal cancer patients, and the fluorescent antibody was dyed and observed with a fluorescent microscope. The imaging results show in Figure 3D that the cell binds to MIL in white light, and there are obvious magnetic nanoparticles around the cell. CK8, 18, 19-FITC has high green-positive fluorescence, DAPI has blue high-positive fluorescence, CD45 is negative, and cells larger than 8 m in diameter can be determined to be CTC. When the tumor size is 50 mm3, 100 mm3, 200 mm3, the CTC count in the outer blood is 4 /mL, 19 /mL, 37 /mL (Figure 3E).

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结果 (英语) 3:[复制]

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result 1. Testing process In this study, peripheral blood samples and corresponding tissue samples of patients with colorectal cancer will be detected. The selected IML preparation process is shown in Figure 1. The antibody reacts with ghdc to form antibody ghdc compound derivative, and then combines with DOPC, cholesterol and fe3o4-hmn to prepare mil by reverse phase one-step method. After obtaining DNA from circulating tumor cells and tissue, the subsequent detection scheme is also shown in Figure 1. By comparing with the data in the public database, we learned that the target miRNA was down regulated in colorectal cancer, designed a series of experiments to verify, and studied the related role of the target miRNA in the occurrence and development of colorectal cancer. Performance evaluation of CTC separation and detection system The microstructure and physicochemical characterization of IMLS are shown in Figure 2. The UV absorption spectrum of mil is shown in Fig. 2A. There are clear UV absorption peaks near 280 nm for EpCAM IML and EGFR IML. The results of UV absorption showed that the antibody was established on the surface of magnetic nanoparticles, and the content of antibody on the surface of magnetic nanoparticles was 0.1mg/mg. It can be seen from the magnetic saturation curve (Fig. 2b) that the prepared microspheres have high saturation magnetization, showing superparamagnetic characteristics, which can be effectively used for the separation of tumor cells. Particle size analyzer was used to detect the change of particle size. The results showed that the modified EpCAM antibody and EGFR antibody were 180 nm and 202 nm, respectively (Fig. 2C). Small particle size is helpful to improve the stability of antibody magnetic liposomes in aqueous solution. The prepared EpCAM and EGFR magnetic beads meet the expected requirements. In addition, the surface potential of epcam-iml and egfr-iml is 0.06 ± 0.02 MV, which is neutral and can reduce the interference of non-specific adsorption caused by positive current reported by previous studies (Fig. 2D). Under AFM, the two kinds of particles have similar spherical shape, but the distribution uniformity is relatively low. The particle diameter is greater than 100 nm, and the particle surface is thicker (Fig. 2e-2f). Target recognition ability of IML on colorectal cancer cells The cytotoxicity analysis of EpCAM mil showed that the survival rate of SW480 and LoVo cells decreased with the prolongation of treatment time at the same concentration of EpCAM mil. When the concentration of EpCAM mil was higher than 200 μ g / ml, the cytotoxicity of EpCAM mil to colorectal cancer cells gradually increased (Fig. 3a-3b). The above results showed that the two IML had the same inhibitory effect on tumor cells, and the inhibitory ability was only related to the concentration of IML. The results of laser scanning confocal microscopy showed that the red fluorescence intensity in the nucleus of SW480 cells was significantly higher than that of Lovo cells, indicating that the expression level of APC in SW480 cells was significantly lower than that in PC-3 cells (Fig. 3C). CTC was isolated from blood samples of patients with colorectal cancer and observed by fluorescence microscope after staining with fluorescent antibody. The imaging results are shown in Fig. 3D. The cells are combined with mil under white light, and there are obvious magnetic nanoparticles around the cells. The fluorescence of fitpi positive cells was higher than that of CDC 8 μ m and CDC 18 μ M. When the tumor size was 50 mm3, 100 mm3 and 200 mm3, the number of CTC in peripheral blood was 4 / ml, 19 / ml and 37 / ml respectively (Fig. 3e).

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