针对RhD血型抗原的单链寡核苷酸适配体修饰红细胞毒性的初步研究  

Preliminary study of ssDNA aptamer cytotoxicity on RhD^(+) red blood cells

作　　者：李璐[1,2] 张印则 王斌 王铸 LI Lu;ZHANG Yinze;WANG Bin;WANG Zhu(Shantou University Medical College,Shantou 515000,China;Shenzhen University General Hospital;The People's Hospital of Longhua,Shenzhen)

机构地区：[1]汕头大学医学院,广东汕头515000 [2]深圳大学总医院 [3]深圳市龙华区人民医院

出　　处：《中国输血杂志》2022年第5期484-487,共4页Chinese Journal of Blood Transfusion

基　　金：国家自然科学基金(81802566);深圳市龙华区科技创新基金项目(2020013、2020003);深圳市南山区卫生科技项目(NS2021164)。

摘　　要：目的 探讨RhD血型抗原的单链寡核苷酸(ssDNA)适配体对红细胞毒性的影响。方法 通过基因合成得到的2条全长82 bp ssDNA的RhD血型抗原适配体;收集5份EDTA抗凝全血,分别制备成4×10^(7)/mL的悬浮红细胞,均分为10管,各5管(100μL/管)标记为实验组:加入2条单特异性ssDNA全长序列(100 pmol/μL)各5μL;对照组:加入等量生理盐水。2组在37℃孵育洗涤60 min后,在LISS液中重悬,于4℃贮存。根据贮存时间不同将实验组和对照组进一步分为1 h、1 d、3 d、10 d、17 d共5个组(每组重复5个血液标本);以FITC标记的Annexin V作为探针标记不同贮存时间LISS悬液中红细胞的磷脂酰丝氨酸(PS),以Fluo-4标记不同贮存时间LISS悬液中红细胞的Ca^(2+)。分别通过流式细胞仪检测不同保存时间LISS悬液中红细胞PS的外翻情况以及红细胞中Ca^(2+)的变化。结果 光学显微镜镜检:孵育后的实验组未发生凝集,对照组发生凝集。流式细胞仪检测:2组间相同贮存时间悬浮红细胞的Annexin V-FITC染色细胞数相近(P>0.05);实验组Annexin V细胞凋亡率(%):贮存10 d时为6.06±1.38,明显高于贮存1 h(P<0.05),贮存17 d时为7.77±1.23,明显高于贮存1 h、1 d、3 d(P<0.05)。2组间在相同贮存时间悬浮红细胞Fluo-4 AM细胞凋亡率相近(P>0.05);实验组Fluo-4 AM细胞凋亡率(%)贮存3 d、10 d、17 d分别为20.84±4.16、22.35±3.37、27.06±2.81(P<0.05)。结论 未发现ssDNA适配体对红细胞存在细胞毒性影响;RhD ssDNA适配体或有望成为检测及制备通用血的材料。Objective To investigate the effect of ssDNA aptamer of RhD blood group antigen on erythrocyte toxicity.Methods Two full-length ssDNA aptamers(82 bp) of RhD blood group antigen were obtained by gene synthesis.Five samples of whole blood with EDTA anticoagulant were collected to prepare red blood cell suspensions(4×10^(7)/mL),which were split into 10 tubes(100 μL/tube),corresponding to 5 experimental groups and 5 controls.Two monospecific full-length ssDNA sequences(100 pmol/μL,5μL each) were added into the experimental group, while the same amount of normal saline into the control.After treatment, the experimental group and the control were incubated for 60 min at 37℃.After washing, they were suspended in LISS solution and stored at 4℃.The experimental group and the control were set according to different time point during storage(1 h, 1 d, 3 d, 10 d and 17 d),with 5 tubes in each group.For erythrocytes in LISS suspension at different storage time, Annexin V labeled with FITC was used as a probe to label the phosphatidylserine(PS) content and Fluo-4 to label Ca^(2+).The eversion of PS and the change of Ca^(2+) concentration in red blood cells in LISS suspensions were determined by flow cytometry.Results After incubation, all groups were examined under the light microscope.No agglutination occurred in the experimental group, while agglutination occurred in the control.Flow cytometry showed that the number of Annexin V-FITC staining cells of suspended erythrocytes at the same storage time-point was similar between the experimental group and the control, with no significant differences.In the experimental group, apoptosis rate of Annexin V cells at 10-day storage(6.06±1.38) was significantly higher than that at 1-hour storage(P<0.05),so as at 17-day storage(7.77±1.23) than 1-hour, 1-day and 3-day storage(P<0.05).The apoptosis rate of Fluo-4 AM cell in suspended RBCs at the same storage time-point was similar between the two groups(P>0.05).In the experimental group, the apoptosis rate of Fluo-4 AM cell at t

关 键 词：单链寡核苷酸(ssDNA) 适配体(NAA) RhD血型抗原 细胞毒性 

分 类 号：R457.11[医药卫生—治疗学] Q524.3[医药卫生—临床医学]