人血小板在WT小鼠和SCID小鼠体内复苏率的比较

Comparison of human platelet recovery between wild type and severe combined immunodeficient mice

目的观察人血小板(hPLTs)输注给WT小鼠和SCID小鼠后,在2种小鼠体内的复苏情况。方法通过一侧尾静脉分别给11只WT(FVB/NJ)小鼠和10只SCID小鼠输注单采的hPLTs 200μl/只,在输注后5、60、120、240 min从WT和SCID小鼠对侧尾静脉,以及360 min从SCID小鼠对侧尾静脉采集小鼠全血10μl/只(次),用3.8%EDTA抗凝后,立即采用4℃保存的1%多聚甲醛固定,4℃过夜保存;将固定的小鼠全血样本经过洗涤和再悬浮后,采用含有磁珠的TruCOUNT试管,用小鼠抗人FITC-CD61抗体和大鼠抗小鼠PE-CD41抗体染色细胞,流式细胞仪定量分析小鼠全血中复苏的hPLTs:以输注后5 min采集的小鼠全血中的hPLTs数量作为基数值,其余时间点的hPLTs数值分别与之比较,计算hPLTs输注后的复苏率。统计学分析比较hPLTs输注后,在WT和SCID小鼠体内的复苏率。结果WT和SCID小鼠体内hPLTs的复苏率,在输注后120 min分别为(37.15±10.39)%和(54.88±18.33)%(t=0.002,P<0.01);在输注后240 min分别为(20.69±8.44)%和(51.07±11.17)%(t=0.000,P<0.01);在输注后360 min在SCID小鼠体内的复苏率为(36.20±7.50)%。结论输注hPLTs后120和240 min,SCID小鼠体内hPLTs的复苏率明显高于WT小鼠,提示SCID小鼠可能作为一种动物模型替代人体实验来评价在体hPLTs的功能。

Objective To observe hPLT recoveries in wild type （WT） and SCID mice after hPLTs transfusion. Methods Two hundred μl of unmodified apheresis hPLT were transfused into WT （FVB/NJ, Jackson Laboratories） and SCID mice through tail veins. Tail crop technique from the other lateral tail veins was employed to collect the mouse whole blood （MWB） at 5, 60, 120, and 240 mins （ 11 WT and 10 SCID mice） and 360 mins （ 10 SCID mice） after transfusion. Five parallel MWB samples of 10 μl were obtained by collecting 5 times from each mouse at each time point. The MWB samples were mixed with 3.8% EDTA. The anticoagulated MWB samples were immediately fixed in cold 1% paraformaldehyde （ 1% paraformaldehyde in PBS） and held overnight at 4℃. Then the fixed MWB samples were washed 3 times and the pellets were re-suspended in wash solution （PBS ＋0. 1% sodium azide ＋ 1% FBS）. hPLT and mouse platelets in the re-suspended MWB samples were double-stained in quadruplicate with PE rat anti-mouse CD41 and FITC mouse anti-human CD61 in TruCOUNT tubes as an internal volume standard. Data were acquired with a flow cytometer. Recoveries of hPLT at different time points after transfusion into the mice were expressed as the percentages of the number of hPLT recovered in circulation at 5 minutes after transfusion. Results hPLT recoveries in WT and SCID mice were （37. 15±10. 39）% and （54. 88 ±18. 33）% （t = 0. 002,P 〈 0.01 ） at 120 minutes after transfusion, respectively, and（20. 69±8.44）% and （ 51.07±11.17 ） % （ t = 0. 000, P 〈 0.01 ） at 240 minutes after transfusion, and the recovery in SCID mice was （ 36. 20±7. 50） % 360 minutes after transfusion. Conclusion The prolonged hPLT existence in the SCID mouse circulation suggests that the SCID mouse model may be employed as an in vivo model to determine the function of human platelets.