脐血巨核细胞体外扩增及其功能的研究

In vitro expansion and function of cord blood megakaryocyte

目的研究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。方法收集足月妊娠健康新生儿的脐带血,免疫磁珠法分离出其中的 CD_(34)^+细胞。采用血小板生长因子(thrombopoietin,TPO)+干细胞因子(stem cell factor,SCF)+白细胞介素3(interleukin-3,IL-3)+IL-6和 TPO+SCF 两种细胞因子组合,将富集的脐血 CD_(34)^+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;血浆块法检测巨核细胞集落(colony forming unit-megakaryocyte,CFU-MK)的形成;对巨核细胞进行 DNA 含量检测以评价其成熟程度;血小板体外活化实验及 SCID 小鼠体内移植实验评价扩增后巨核细胞的功能。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(CD_(41)^+)的数量逐渐增加,但培养至14 d 时增势减缓。因子组合TPO+SCF+IL-3+IL-6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TPO+SCF(分别扩增3.8、27.4、85.9、106.5倍)强,但因子组合 TPO+SCF 的扩增能力仍能满足临床的需要。巨核祖细胞(CD_(34)^+CD_(41)^+)的数量在第7天时最多(分别增加43.4和36.2倍),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。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,有正常巨核细胞功能。移植后两组小鼠的骨髓中均检测到人 CD_(45)^+和 CD_(41)^+细胞。小鼠外周血中人血小板在移植后3 d 就可测到,5 d 就可达到高水平(分别为20.7%和17.9%),维持20 d 以后才逐渐下降。结论通过对扩增后巨核细胞的生物学特性的研究,有助于寻找有效、简便、易于植入受者体内的扩增方法。体外扩增的脐血巨核细胞可植入骨髓并产生功能正常的血小板。

Objective Cord blood （CB） provides a rich source of stem ceils for transplantation. CB transplantation has been used widely after myeloablative therapy. One major disadvantage of CB transplantation is delayed platelet engraftment. The aim of this study was to hasten platelet engraftment by investigating the ability of different hematopoietic growth factor combinations to generate large numbers of megakaryocyte （Mk） from CB and by evaluating the biologic characteristics and function of the expanded Mk. Methods CB samples were obtained at the end of normal full-term deliveries with informed consent. Mononuclear ceils （MNCs） were isolated from CB using Ficoll density centrifugation. MNC population was positively selected for CD34 expression by magnetic cell sorting （MACS）. CD34^＋ cells were 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 characterized after 3, 7, 10 and 14 days by flow cytometry, colony forming unit-megakaryocyte （CFU-Mk） and maturation evaluation （Mk ploidy）. The expanded Mk function was examined by the platelet activation in vitro and severe combined immunodiffiency （SCID） mice transplantation in vivo. Results Different results were observed with different culture conditions. With the first cytokine combination optimal expansion of CD41^＋ cells was observed on day 10, but the optimal expansion of Mk progenitors （CD34^＋CD41^＋） was observed on day 7, with a median 121 and 44-fold increase at the starting cell dose. This result was also proven by CFU-Mk. The largest numbers of CFU-Mk were also observed on day 7. The degree of maturation of Mk cells also increased as suggested by DNA content of CD41^＋ cells, which means that CD34^＋ cells cultured for 3-7 days were richer in primitive Mks, while those cultured for 10-14 days had greater numbers of more differentiated Mks. For the second cytokine combination, CD41^＋ and CD34^＋CD41^＋ cells were fewer than the first one, but it produced 36 and 85-fold CD34^＋CD41^＋ and CD41^＋ respectively on day 7. Platelet activation test confirmed that the expanded Mks had normal function. Therefore, the expanded Mks could be transplanted into the SCID mice bone marrow and produce human platelet in the peripheral blood of the mice. Conclusion Ex vivo expanded Mk might facilitate CB transplantation and help shorten the period of post-transplant thrombocytopenia.