IL-6/sIL-6R对人脐血CD34+细胞体外扩增的作用

背景和目的:造血干细胞具有自我更新、多向分化与重建长期造血的潜能,因此,造血干细胞广泛应用于干细胞移植、免疫治疗、基因治疗等领域.胎儿脐带血中富含造血干细胞,但单份脐带血中造血干细胞的数量有限,不能充分满足临床和科研需要,因此在体外培养使脐带血干/祖细胞数量扩增的研究日益受到重视.已知一些细胞因子可以在体外培养中使脐血干/祖细胞大量扩增.新近发现IL-6/sIL-6R(可溶性IL-6受体)或其融合蛋白可促使脐带血CD34+细胞中CD34+gp130+IL-6R-细胞亚群在体外培养中大量扩增.本实验旨在观察IL-6/sIL-6R在脐带血CD34+细胞体外扩增中的作用,并探讨合适细胞因子组合.方法:脐血CD34+细胞用Mini MACS分离,然后在含有不同细胞因子组合的液体培养基中体外培养7天或14天,培养前后分别进行有核细胞计数、用FCM(流式细胞术)测定CD34+细胞比例计算CD34+细胞总数及进行CFU-GM集落培养.根据不同细胞因子组合分为空白对照组,SCF组,SCF+IL-6/sIL-6R组,SCF+FL+IL-6/sIL-6R组,SCF+FL组.结果:空白对照组和SCF组CD34+细胞数量在培养7天或14天后明显下降.SCF+IL-6/sIL-6R组培养7、14天分别使有核细胞及CD34+细胞绝对数扩增(7.1±2.4)倍、(39.0±14.0)倍及(1.8±0.7)倍、(4.8±2.4)倍;SCF+FL+IL-6/sIL-6R组(16.5±5.7)倍、(110.0±28.0)倍及(3.5±1.5)倍、(10.2±4.2)倍;SCF+FL组(17.3±3.8)倍、(104.0±21.0)倍及(3.6±2.1)倍、(8.4±3.5)倍.上述三组与对照组及SCF组差异均有显著性(P＜0.01),其中SCF+FL+IL-6/sIL-6R组和SCF+FL组扩增效果优于SCF+IL-6/sIL-6R组(P＜0.01),但是SCF+FL+IL-6/sIL-6R组和SCF+FL组之间差异无显著性(P＞0.05).增加sIL-6R浓度,当sIL-6R浓度为400 ng/ml时,细胞培养7天,SCF+FL+IL-6/sIL-6R组分别使有核细胞及CD34+细胞绝对数扩增(24.0±4.8)倍、(5.6±1.2)倍,优于SCF+FL组(P＜0.05).结论:IL-6/sIL-6R可以和SCF、FL产生协同作用,使人脐血CD34+细胞在体外扩增、并维持其分化潜能.但这种协同作用对sIL-6R的浓度存在一定依赖性.

Effect of IL-6/sIL-6R on ex vivo expansion of human cord blood derived CD34+ cells

BACKGROUND & OBJECTIVE:Hematopoietic stem cells (HSC) have the ability of regeneration, differentiation, and reconstructing hematopoietic function, and it is widely used in many fields such as hematopoietic stem cell transplantation, immune therapy, gene therapy and so on. Human cord blood (CB) is abundant of HSC. But a single collection of CB has only a limited amount of HCS and cannot fit the clinical and research use. Thus ex vivo expansion of human CB derived HSC is important. We know that there are some cytokines, which can synergize for enhancing the expansion of CB derived CD34(+) cells in vitro. Currently, some experiments have discovered that IL-6/sIL-6R or its chimera can enhance the ex vivo expansion of CD34(+)gp130+IL-6R- subpopulation. This study was designed to observe the effect of IL-6/sIL-6R on the ex vivo expansion of human CB derived CD34(+) cells, and explore the optimal cytokine combinations. METHODS: Human CB derived CD34(+) cells were isolated by Mini MACS and cultured in ex vivo liquid media in the presence of different cytokine cocktails for 7 or 14 days. After cultured on the seventh or the fourteenth day, the total number of the cultured cells were counted, the ratio of the CD34(+) cell were assayed by flow cytometry (FCM) and the number of it were calculated, and CFU-GM were cultured, then the effects of different cytokine combinations on the ex vivo expansion of CD34(+) cells were compared. In line with the different cytokine cocktails, our experiment divided into five groups: (A) control,(B) SCF,(C) IL-6/sIL-6R+SCF,(D) IL-6/sIL-6R+SCF+FL,and (E) SCF+FL. RESULTS: After cultured in vitro for 7 or 14 days, (1) the number of CD34(+) cells descended apparently in groups A and B; (2) the number of nucleated cells and CD34(+) cells after cultural on the seventh or the fourteenth day increased 7.1+/-2.4 folds, 39.0+/-14.0 folds; 1.8+/-0.7 folds, 4.8+/-2.4 folds, respectively in group C; 16.5+/-5.7 folds, 110.0+/-28.0 folds; 3.5+/-1.5 folds, 10.2+/-4.2 folds in Group D; 17.3+/-3.8 folds, 104.0+/-21.0 folds; 3.6+/-2.1folds, 8.4+/-3.5 folds in Group E. The expansion effects of group C, D, and E were all superior to the group A or B (P< 0.01). The expansion effects of group D and E were superior to group C (P< 0.01). But there was no difference between group D and E (P >0.05); (3) Adding the concentration of sIL-6R to 400 ng/ml, the number of nucleated and CD34(+) cells increased 24.0+/-4.8 folds and 5.6+/-1.2 folds in group D after cultured for seven days superior to group E (P< 0.05). CONCLUSION: IL-6/sIL-6R, SCF, FL can synergize for enhancing the ex vivo expansion of human CB derived CD34(+) cells. But this synergetic effect depends on the concentration of sIL-6R.