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目的验证ssDNA适配体阻断RhD抗原-抗体的结合,进而阻断FcγR介导巨噬细胞吞噬作用的能力。方法0.2%红细胞悬液分为三组:实验组(加入经ssDNA处理过的RhD IgG单克隆抗体)、空白对照(未处理的红细胞悬液)、阳性对照(加入未经ssDNA处理的RhD IgG单克隆抗体)。37℃孵育30 min。洗涤后加入1∶500的羊抗人IgG Alexa Fluor 488荧光抗体,37℃,30 min。洗涤后流式细胞仪检测。单核细胞单层试验收集6人份外周血单核细胞于37℃混合培养1h,洗去未贴壁细胞。以2∶1体积加入不含荧光抗体的三组红细胞,培养1.5 h后进行瑞氏-姬姆萨染色。镜下观察500个单核细胞,计算平均吞噬指数。结果流式结果显示,阳性对照荧光强度为3 166.33±172.55,而实验组荧光强度仅为307.00±45.18,与空白对照组(287.33±30.50)无显著性差异。MMA实验结果显示,阳性对照组单核细胞平均吞噬指数(7.20±1.48)Cut-off值(0.35),结果为阳性;试验组的吞噬指数(0.07±0.12)低于Cut-off值,结果为阴性。结论 ssDNA与RhD抗体结合后可使RhD抗体丧失与RhD抗原特异性结合的能力,以至于红细胞不会被致敏,为新生儿溶血病的治疗提供了新的思路。
Objectives To verify that ssDNA aptamers block the binding of RhD antigen-antibodies, thereby blocking the ability of FcyRs to mediate macrophage phagocytosis. Methods 0.2% erythrocyte suspensions were divided into three groups: experimental group (rhodo-IgG monoclonal antibody treated with ssDNA), blank control (untreated erythrocyte suspension), positive control (treated with ssDNA-treated RhD IgG Clone antibody). Incubate at 37 ° C for 30 min. After washing, 1: 500 goat anti-human IgG Alexa Fluor 488 fluorescent antibody was added and incubated at 37 ° C for 30 min. After washing flow cytometry. Monocyte monolayer test 6 human peripheral blood mononuclear cells were collected and mixed at 37 ℃ for 1 hour, washing off the non-adherent cells. Three groups of erythrocytes without fluorescent antibody were added in a volume of 2: 1, and Wright-Giemsa staining was performed after culturing for 1.5 h. 500 monocytes were observed microscopically and the average phagocytic index was calculated. Results The results of flow cytometry showed that the fluorescence intensity of the positive control was 3 166.33 ± 172.55, while the fluorescence intensity of the experimental group was only 307.00 ± 45.18, which was not significantly different from that of the blank control group (287.33 ± 30.50). The results of MMA showed that the positive phagocytosis index (7.20 ± 1.48) Cut-off value (0.35) of monocytes in the positive control group was positive, and the phagocytic index (0.07 ± 0.12) in the experimental group was lower than the Cut-off value. The results Negative. Conclusion The combination of ssDNA and RhD antibody can abolish the ability of RhD antibody to bind specifically with RhD antigen so that erythrocytes will not be sensitized and provide a new idea for the treatment of hemolytic disease in neonates.