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体外研究表明,人脐血含有比骨髓细胞更原始更早期的造血干细胞群。移植后所引起的GVHD发生率及程度都比骨髓及外周血要低,但其相应的GVL效应也较低,容易复发。单份脐血所含有的造血细胞量仅可以满足一定体重以下的儿童患者需求,对需移植的大部分成人患者则要进行体外扩增。脐血体外扩增后进行干细胞移植是一种新的设想和尝试,移植物中免疫细胞的组成和功能是决定干细胞移植能否成功重建造血与免疫、以及平衡GVHD和GVL的重要因素。为了研究脐血体外同时诱导扩增T、NK和CD34+细胞的可能性,本实验无菌采集健康正常足月产新生儿脐血,并分离出单个核细胞,在不同的细胞因子组合作用下用IMDM培养液培养14天。在培养0、3、7、14天时收集细胞,用FCM分析扩增前后脐血干/祖细胞及T、NK免疫细胞含量。结果表明,与无细胞因子的对照组相比,所有细胞因子SCF,IL3,IL6,IL7,IL2组合组别均能显著扩增脐血中的单个核细胞。所有细胞因子组合组别均能显著增加脐血中的CD34+细胞比例,使其含量从新鲜脐血中的1.6%升高到最高D组的11.9%。第7天时CD34+细胞平均增加10至50倍不等。新鲜脐血中CD3+T细胞平均为(18.7±4.3)%,在无细胞因子的对照组中CD3+T细胞下降明显,而在含细胞因子的组别中CD3+T有不同程度的增加,扩增最高的组别中CD3+T细胞是新鲜脐血的2倍。在新鲜脐血中含(3.6±1.9)%CD56+细胞。CD56+细胞数量仅在含细胞因子IL2的组别中有显著增加,其它组则无明显变化。结论:脐血中T细胞、NK细胞在一定细胞因子组合下,可与干/祖细胞同时在体外被扩增和诱导分化。
In vitro studies have shown that human umbilical cord blood contains more primitive hematopoietic stem cell populations than bone marrow cells. After transplantation, the incidence and extent of GVHD are lower than that in bone marrow and peripheral blood, but the corresponding GVL effect is also low and relapse easily. The amount of hematopoietic cells contained in a single cord blood can only meet the needs of children under a certain weight, and the majority of adult patients who need to be transplanted have to undergo in vitro expansion. The expansion of umbilical cord blood after stem cell transplantation is a new idea and attempt, the composition and function of immune cells in the graft is to determine the successful reconstruction of hematopoietic and immune stem cell transplantation and an important factor in the balance of GVHD and GVL. In order to study the possibility of simultaneously inducing the expansion of T, NK and CD34 + cells in cord blood in vitro, cord blood of healthy full-term full-term newborns was collected aseptically in this experiment and mononuclear cells were isolated. Under different combinations of cytokines IMDM culture medium for 14 days. The cells were harvested at day 0, day 3, day 7, day 14, and the cord blood stem / progenitor cells and T, NK immune cells were analyzed by FCM. The results showed that compared with the control group without cytokines, all of the cytokines SCF, IL3, IL6, IL7, IL2 combination group can significantly amplify cord blood mononuclear cells. All combinations of cytokines significantly increased the proportion of CD34 + cells in cord blood, increasing their levels from 1.6% in fresh cord blood to 11.9% in those in the highest D group. On day 7, CD34 + cells averaged an increase of 10 to 50 fold. The average number of CD3 + T cells in fresh cord blood was (18.7 ± 4.3)%. CD3 + T cells decreased significantly in the control group without cytokines, while CD3 + T increased in different groups with cytokines, CD3 + T cells in the highest group were twice as fast as fresh cord blood. In fresh cord blood (3.6 ± 1.9)% CD56 + cells. The number of CD56 + cells increased only significantly in the group containing IL2 but not in other groups. Conclusion: T cells and NK cells in umbilical cord blood can be expanded and differentiated in vitro with stem / progenitor cells under certain combination of cytokines.