Detection of N-ethyl-N-nitrosourea-induced Pig-a gene mutation in rats by immunomagnetic separation technique combined with flow cytometry
Received:December 02, 2017  
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DOI:10.3969/j. issn. 1005 -4847. 2018. 02. 012
KeyWord:Pig-a gene mutation; immunomagnetic separation; flow cytometry; N-ethyl-N-nitrosourea; ENU;
秦美蓉 深圳市药品检验研究院,深圳药品质量标准研究重点实验室,广东深圳
郑丹丹 深圳市药品检验研究院,深圳药品质量标准研究重点实验室,广东深圳
黄泽愉 深圳市药品检验研究院,深圳药品质量标准研究重点实验室,广东深圳
王晓炜 深圳市药品检验研究院,深圳药品质量标准研究重点实验室,广东深圳
李俊鹏 深圳市药品检验研究院,深圳药品质量标准研究重点实验室,广东深圳
王平 深圳市药品检验研究院,深圳药品质量标准研究重点实验室,广东深圳
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      Objective To optimize the detection test of Pig-a gene mutation in peripheral blood of rats by enriching and detecting mutant erythrocytes, using immunomagnetic separation technique in combination with flow cytometry. Methods SD rats were administered with 20, 40 and 80 mg/ (kg·bw) doses of N-ethyl-N-nitrosourea (ENU) continually for 3 days. The peripheral blood samples of rats were collected on the 7th, 14th and 28th days, respectively, after treatment. Immunomagnetic separation columns were used to enrich RET CD59 - and RBC CD59- cells, and then flow cytometry was used to count the number of pre-column and post-column peripheral erythrocytes. Results Compared with the control group, the frequencies of RET CD59- and RBC CD59- were significantly increased in each ENU group ( P <0.05). With immunomagnetic separation technique, the test of Pig-a gene mutation of a sample could be completed within 3 minutes, and the number of detected RET CD59 - or RBC CD59 - cells was up to 2 ×10 4 or 9 ×10 4 , respectively. Conclusions In this study, immunomagnetic separation in combination with flow cytometry is used to establish and optimize the Pig-a gene mutation test in rat peripheral blood, showing a high-throughput detection and improved accuracy and efficiency of the experiment.
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