尿素干预膜KCC转运体对有核红细胞富集的实验研究

Study on the enrichment of nucleated red blood cells through interfering membrane KCC by urea

目的通过对尿素干预K-Cl同向转运体(K+/Cl-cotransporter,KCC)对红细胞(RBC)容积改变的观察,探讨建立有效富集分离有核红细胞(NRBC)方法。方法测量在不同渗透压环境下、不同尿素浓度和作用时间后脐血RBC直径,建立改变细胞容积的最佳干预条件。流式细胞仪观察最佳条件下尿素干预脐血KCC转运体后富集NRBC的效果。结果尿素浓度、渗透压条件及时间变化3种干预条件之间有交互作用(F=26.729,P<0.05)。在400 mM高渗环境下尿素干预10 min后可以使成熟RBC体积变小最明显(t=2.62,P<0.05)。干预脐血后1.065 g/mL密度介质离心下层RBC白膜层FNRBC富集量由12.2%升为69.9%(u=7.96,P<0.05),单个核上层FNRBC由7.15%降为2.00%(u=5.47,P<0.05)。结论尿素干预脐血可以增加NRBC富集率达69.9%,较传统富集法增加了约5倍的富集量。

Objective To establish an effective method for separating and enriching nucleated red blood cell（RBC）（NRBC） with observing the change of RBC volume through interfering K＋/Cl-cotransporter（KCC） by urea.Methods The intervention condition of maximal change of cell volume was constructed by observing mature RBC diameter after interfering cord blood with different action time and urea concentration（400 mM,200 mM and 100 mM） and different osmotic pressure.Under this intervention condition,the effect of NRBC′s enrichment through interfering KCC by urea was observed by flow cytometry.Results There was an interaction among the intervention conditions of urea concentration,osmotic pressure and time change（F=26.729,P0.05）.The volume of mature RBC decreased after intervention of both high osmotic pressure and 400 mM urea for 10 min（t=2.62,P0.05）.Under 1.065 g/mL centrifugation medium,the NRBC enrichment of underlayer white film increased from 12.2% to 69.9%（u=7.96,P0.05） after the urea intervention.The NRBC enrichment of upper white film reduced from 7.15% to 2.00%（u=5.47,P0.05）.Conclusions The urea intervention can promote the rate of NRBC′s enrichment to 69.9%,and it is about 5 times than traditional methods.