豆类凝集素FRIL通过调控细胞周期分子的表达维持造血干/祖细胞的静息

目的从细胞周期角度探讨豆类凝集素 FRIL(Flt3 receptor-interacting lectin)体外维持造血干/祖细胞静息的分子机制。方法以添加 FRIL、Flt3配体(FL)和不添加因子的培养基分别培养脐血 CD34~ 细胞,采用 RT-PCR 和 Western blot 法分别从 mRNA 和蛋白水平检测细胞周期相关分子的表达。结果新分离的 CD34~ 细胞中未检测到 G_0/G_1期相关 Cyclin 和 CDK 蛋白的表达,培养3d 时FRIL 组 CyclinD3、CDK6蛋白相对表达量(分别为483±63、553±39)低于两个对照组(FL 组:2437±52、3209±98;空白对照组:914±105、1497±55);培养3 d 时 FRIL 组 P27蛋白相对表达量(0.312±0.030)低于对照组(FL 组:0.787±0.024;空白对照组:0.616±0.029),但表达较高的 P53蛋白(FRIL组、FL 组、空白对照组分别为4.476±0.159、0.581±0.099、2.167±0.114)。FRIL 组细胞周期正向调节因子的 mRNA 相对表达量低于或相当于 FL 组和空白对照组。结论 FRIL 通过抑制参与造血细胞周期调控的 CyclinD3和 CDK6的表达,延迟了造血干/祖细胞的细胞周期,P27在 FRIL 抑制造血干/祖细胞分化中发挥了重要作用,P53也参与了 FRIL 对造血干/祖细胞的维持作用。

FRIL maintains quiescence of hematopoietic stem cells through regulation of cell cycle related factors

OBJECTIVE: To explore the mechanism of Flt3 receptor-interacting lectin (FRIL) maintains quiescence of hematopoietic stem cells (HSCs) in vitro. METHODS: Cord blood CD34+ cells were cultured in suspension medium supplemented with or without FRIL and FL. Cells were collected at different time points and the expression of some cell cycle regulators, especially those involved in G0/G1 phase regulation were detected on mRNA and protein level. RESULTS: The expressions of G0/G1 phase related cyclins or CDKs were undetectable in the newly isolated CD34+ cells, expressions of CyclinD3, CDK6 and P27 were the lowest in FRIL cultured group after 3d's culture (FRIL group: 483 +/- 63, 553 +/- 39, 0.312 +/- 0.030; FL group: 2437 +/- 52, 3209 +/- 98, 0.787 +/- 0.024; BLANK: 914 +/- 105, 1497 +/- 55, 0.616 +/- 0.029, respectively), but the expression of P53 was the highest in FRIL group (FRIL group: 4.476 +/- 0.159; FL group: 0.581 +/- 0.099, BLANK: 2.167 +/- 0.114). The expression of positive regulators of cell cycle in FRIL group were the same as that of FL group and blank group or lower. CONCLUSION: FRIL preserves HSCs effectively in vitro through the mechanisms of down-regulation of cyclinD3 and CDK6 and activation of P53. P27 is mostly involved in the differentiation of HSCs.