重组人脱氧核糖核酸酶抑制冻存复苏后脐血造血干细胞凝集

Recombinant human deoxyribonuclease suppressing cell agglutination of thawed and processed cord blood henmtopoietic progenitors

目的探讨重组人脱氧核糖核酸酶（rhDNase）抑制冻存复苏脐血干细胞微凝集及凝块形成的作用。方法分别将冻存2周、1个月、2个月、半年、1年及2年的脐血干细胞进行复苏，然后用不同浓度（终浓度分别为0、1、5、8、10、20及50U／m1）的rhDNase缓冲液进行洗涤，在显微镜下观察凝块及微凝集的形成情况。采用免疫磁珠法分选CD34^＋细胞，分别经10U／m1和50U／ml的rhDNase缓冲液处理，采用免疫荧光直接四色标记和流式细胞术检测CD34^＋细胞表面CD54、CD11a、CD62L的表达；采用自动血细胞分析仪测定细胞回收率，台盼蓝着色法判断细胞活性，分析10U／ml rhDNase缓冲液洗涤对复苏脐血细胞回收率、细胞活性及凝集率的影响。结果复苏的脐血干细胞经0、1、5、8U／ml的rhDNase缓冲液洗涤后仍有不同程度的凝集；经浓度为10、20、50U／ml的rhDNase缓冲液处理后，没有出现凝块和微凝集。经10U／ml及50U／ml的rhDNase处理后，CD34^＋细胞表面粘附分子的表达无明显改变，与阴性对照相比，差异无统计学意义。经10U／ml的rhDNase缓冲液洗涤后，干细胞回收率有所下降（P〈0．05），但对细胞活性无明显影响，未经洗涤的脐血均有凝集，而洗后均无凝集。结论浓度为10U／ml的rhDNase足以抑制复苏后脐血干细胞凝块和微凝集的形成，且不影响脐血干细胞的活性及粘附分子的表达。

Objective To explore the inhibitory effect of recombinant human deoxyribonuclease on clotting phenomena and microaggregate formation of thawed and processed cord blood hematopoietic progenitors. Methods Six UCB samples which were cryopreserved for 2 weeks, 1 month, 2 months, half a year, 1 year and two years were thawed respectively. Each sample was washed by rhDNase buffer solution in various concentration （final concentration was 0, 1, 5, 8, 10, 20 and 50 U/ml）, and the clot and microaggregates formation were observed under a microscope, CD34^＋ cells were isolated from cord blood by magnetic cell sorting system （MACS） and then incubated with 10 U/ml, 50 U/ml rhDNase, The expression levels of CD54, CDlla and CD62L in CD34^＋ cell were assessed using a highly sensitive 4-color flow cytometric analysis. Mononuclear cells （MNC） were detected by blood cell analyzer. The survival rate was determined by Trypan blue staining. The recovery rate （%） of MNC, viable rate （%） of NC and microaggregate rate （%） of washed samples by rhDNase 10 U/ml was compared with those of unwashed samples respectively. Results Cell clots could be observed at concentrations of 0, 1, 5, 8 U/ml rhDNase, but clotting and microaggregate formation could be prevented for all tested samples at 10 U/ml, 20U/ml and 50 U/ml rhDNase. There was no significant difference in the expression of adhesion molecules between UCB cells treated with rhDNase and untreated cells. Compared with untreated samples, the recovery rate of MNC in treated samples was decreased slightly, while viable rate （%） in NC did not show any significant difference. Cell clots could be observed in all unwashed samples, but could not be found in washed samples. The effect of 10 U/ml rhDNase was identical on various term storage cord blood. Conclusion The concentration of 10 U/ml rhDNase was enough to prevent cell clotting or microaggregate formation and did not influence the expression of adhesion molecules.