体外扩增脐血巨核细胞生物学特性的研究

目的:研究脐血巨核细胞的体外扩增及扩增后脐血巨核细胞的生物学特性和功能,为脐血巨核细胞的扩增和临床应用提供依据。方法:收集16份足月妊娠健康新生儿的脐带血,免疫磁珠法分离出其中的CD34 细胞。采用细胞因子组合(1)(TPO加SCF加IL-3加IL-6)、细胞因子组合(2)(TPO加SCF)2种细胞因子组合．将富集的脐血CD34 接种于无血清无基质细胞的悬浮培养体系中,分别在3、7、10、14 d收集扩增产物。运用流式细胞术检测巨核细胞的表型和黏附分子的表达;血浆块法检测巨核细胞集落(CFU-MK)的形成;对巨核细胞进行DNA含量检测以评价巨核细胞的成熟程度。结果:不同细胞因子组合和培养时间扩增后,巨核细胞的数量和生物学特性不同。随着培养时间的延长,巨核细胞(CD41 )的数量逐渐增加,但培养至14 d时增势减缓。因子组合(1)各时间段的扩增能力(分别扩增5．2、40．7、121．2、149．7倍)均比因子组合(2)(分别扩增3．8、27．4、85．9、106．5倍)强(P<0．05),但因子组合(2)的扩增能力仍能满足临床的需要。巨核祖细胞(CD34 ／CD41 )的数量在7 d时最多,这也被CFU-MK所证实。DNA含量检测发现．随着培养天数的增加,多倍体细胞所占的百分比增加。扩增后巨核细胞黏附分子CD49d和CD11a的表达维持在高水平,而CD54降低(P<0．05)。结论:通过对扩增后巨核细胞的生物学特性研究,有助于寻找有效、简便、易于植入受者体内的扩增方法。

Biologic chracteristics of ex vivo expanded cord blood megakaryocyte

Objective:To investigate the potential of megakaryocyte(MK) expansion in vitro from cord blood (CB) and the biologic characteristics of ex vivo expanded MK, and in order to provide some bases for the in vitro expansion and clinical application of MK. Method: 16 samples were taken from the umbilical cord of full-term neo-nates. CD34 cells were isolated from CB by immunomagnetic sorting and cultured in serum-free and stroma-free medium containing the following two different cytokine combinations: thrombopoietin(TPO) stem cell factor (SCF) interleukin(IL)-3 IL-6 and TPO SCF. Cultures were gathered after 3, 7, 10 and 14 days. Phenotype and homing-related adhesion molecules(CAMs) of MK were detected by flow cytometry(FCM) ; Colony forming unit-megakaryocyte(CFU-MK) was detected by plasma clotting assay; The MK maturation (MK ploidy) was e-valuated by DNA concentrations analysis. Result: Different results were observed with different culture condition. With the time of culture, expanded CD41 cells gradually increased and became decrease at day 14. The potential of MK expansion with the first cytokine combination was significant higher than that with the second cytokine combination at different period of time ( P <0. 05), while the potential of MK expansion with the latter combination could be enough for clinical use. The optimal expansion of MK progenitors (CD34/CD41) was observed at day 7 and the result was also certified by CFU-MK. The percentage of MK ploidy gradually increased with the time of culture according to the results of DNA concentrations analysis. After MK expansion, the expression of CD49d and CD11a on MK kept at high level, but CD54 decreased significantly ( P <0. 05). Conclusion:The method of expansion is helpful for cord blood transplantation which makes engraftment more effectively, simply and easily by investigating the biological characteristics of expanded MK.