长期液氮保存PBSC细胞凋亡及死亡检测

目的比较外周血干细胞（peripheral blood stem cell，PBSC）采集物中有核细胞经长期深低温保存后细胞凋亡及死亡率与新鲜采集物的差异，便于对PBSC进行活力评价。方法47份外周血干细胞采集物标本，新鲜对照标本12份，实验Ⅰ组为液氮保存2～4年的PBSC26份，实验Ⅱ组为液氮保存5～7年的PBSC9份，以流式细胞仪检测各组有核细胞中核酸染料7AAD着色的细胞百分比（代表死亡率），以及有核细胞中表达活化的caspase-3的细胞百分比（代表凋亡率）。结果两实验组的细胞死亡率和凋亡率均高于对照组（P〈0．05），实验Ⅱ组凋亡率为35．6％±17．51％，与实验Ⅰ组（24．14％±16．87％）的差异有显著性（P=0．031）。结论深低温保存2～7年的PBSC采集物中有核细胞活性较新鲜PBSC采集物低，凋亡率及死亡率升高。

Detect to Necrosis and Apoptosis of PBSC for Long-term Cryopreserred by Liquefaction of Nitrogen

Objective To compare nucleated cell viability between the fresh and the long-term cryopreserved PBSC collections,to assist the judgment on the activity of PBSC. Methods Among the total 47 samples of PBSC collections, frozen samples were divided into two groups： group 1（cryopreserred for 2-4 years,n=26） and group 2 （cryopreservd for 5-7 years,n=9） ;fresh samples （n=12） were taken as control. Flow cytometry method （FCM） based on 7AAD or caspase-3 staining was applied to assess the degree of cell necrosis （7AAD＋） or apoptosis（caspase-3＋） in cryopreserved and red-cell lytic collection samples. Results Both the necrosis and apoptosis levels of group 1 and group 2 were higher than those of control group（P〈0.05）; apoptosis cell of group 2 was more than that of group 1 （35.6%±17.51% to 24.14± 16. 87% ,P = 0. 031）. Conclusion Compared with the fresh PBSC collections, the long-term frozend ones had decreased NCs viabilities.