脐血巨核祖细胞的体外扩增

In Vitro Expansion of Cord Blood Megakaryocyte Progenitor

目的联合血小板生成素(TPO)、白细胞介素-11(IL-11)和肝素用脐血CD34+细胞定向扩增巨核祖细胞。方法采用免疫磁珠法(M ACS)分选CD34+细胞,用TPO、IL-11和肝素定向扩增巨核祖细胞,巨核祖细胞集落分析(CFU-M K)测定巨核祖细胞扩增倍数,流式细胞术检测巨核祖细胞分化过程中不同细胞组群(CD34+、CD41a+、CD61+、CD34+CD41a+和CD41a+CD61+)的变化,免疫组织化学染色(CD41a)和透射电镜观察巨核细胞形态及超微结构,血小板体外活化实验及非肥胖性糖尿病/严重联合免疫缺陷鼠异种体内移植实验评价扩增的巨核祖细胞功能。结果单用TPO7d时,CD34+CD41a+细胞扩增(4.0±1.7)倍;与IL-11联用后扩增到(10.5±4.8)倍;再加入肝素后扩增达(29.9±6.4)倍,TPO+IL-11+肝素组扩增倍数为TPO组、TPO+IL-11组的7.5、2.85倍,与两组相比差异均有显著性(P<0.05)。TPO+IL-11+肝素组巨核祖细胞中大集落(>50个细胞/集落)达(106.8±26.9)倍,较TPO、TPO+IL-11组均有明显增加(P<0.05)。将扩增第7天的巨核细胞静脉输注于经放射预处理的非肥胖性糖尿病/严重联合免疫缺陷鼠,可明显加速其血小板及白细胞的恢复并提高小鼠生存率。电镜显示扩增的巨核细胞有一定的界膜发育等成熟特征,体外血小板活化实验证实,扩增的巨核细胞在体外可产生血小板。

Objective To expand cord blood megakaryocyte progenitor cells in vitro. Methods Cord blood CD34+ cells were selected by magnetic cell sorting(MACS), and thrombopoietin(TPO), interleukin-11(IL-11), and heparin were used in the expansion system of megakaryocyte progenitor. The expansion efficiency was measured by fluorescence-activated cell sorting(FACS)using the megakaryocytic specific monoclonal antibodies(CD34+, CD41a+, CD61+, CD34+CD41a+, CD41a+CD61+)and colony-forming units-megakaryocyte(CFU-MK)analysis. The expanded megakaryocyte progenitor were determined by histochemistry staining using CD41a and the observation of the ultrastructure of megakaryocyte(MK)by electron microscopy. The megakaryocyte function were examined by the platelet activation in vitro and nonobese diabetic/severe combined immunodifficiency(NOD/SCID)mice transplantation in vivo. ResultsCD34+CD41a+ cells was expanded(4.0 ± 1.7)folds on day 7 in TPO(50 ng/ml)group and(10.5 ± 4.8)fold in TPO combined with IL-11 group; after heparin was joined in on day 0, a more significantly elevated expansion was found in the heparin, TPO, and IL-11 group(29.9 ± 6.4)folds than the above two groups; P < 0.05. Meanwhile, the large CFU-MK colony(> 50 cells/colony)was(106.8 ± 26.9)folds on day 7(P < 0.05). The megakaryocyte expanding with TPO, IL-11 and heparin for 7 days in vitro transplanted the NOD/SCID mice fasten the recovery of platelet and white blood cell account and improved the survival. Megakaryocyte under culture displayed certain development of territories membrane. Platelet activation test comfirmed that the expanding megakaryocyte progenitor had the normal function. Conclusion TPO, IL-11, and heparin combination system for ex vivo expansion is an effective expan-sion system of megakaryocyte progenitor.