Wnt、Notch信号参与造血微环境调控造血机制的研究

目的 探讨造血发育过程中Wnt和Notch信号通路参与造血微环境调控造血的机制.方法 磁珠分选法分离小鼠骨髓c-kit~+造血干/祖细胞(HSPC).贴壁培养法分离小鼠孕10.5 d主动脉-中肾-肾上腺(AGM)区,孕12.5 d、14.5 d、16.5 d胎肝以及骨髓来源的基质细胞.通过Transwell共培养以及流式细胞术计数,比较不同来源基质细胞支持HSPC增殖的能力.通过实时定量PCR和免疫荧光动态检测各发育阶段基质细胞中Wnt、Notch Mrna和蛋白的表达.结果 体外培养7 d,AGM区和胎肝基质细胞培养体系中c-kit~+细胞数由0.4×10~5/孔分别扩增为(19.2±3.2)×10~5/孔和(26.8±5.4)×10~5/孔,明显高于骨髓基质细胞培养体系的(9.6 ± 2.9)×10~5/孔(P＜0.05);其中,AGM区和骨髓基质细胞培养体系中c-kit~+细胞比例分别为(75.2 ± 7.1)%和(74.1 ± 6.2)%,明显高于胎肝基质细胞培养体系的(63.4±5.3)%(P＜0.05).Wnt、Notch在各阶段基质细胞均有表达,Wnt在AGM区和胎肝基质细胞表达水平较高,Notch在AGM区及骨髓基质细胞表达水平较高(P＜0.05).结论 造血发育过程中,造血微环境通过Wnt与Notch信号通路协作、交替、整合,精密调控HSPC的发育.

Study on Wnt and Notch signalling involves in regulation of hematopoietic microenvironment

Objective To explore the mechanism of Wnt and Notch pathway involved modulating time and spatial restricted hematopoiesis. Methods Murine hematopoietic stem and progenitor cells (HSPCs) were isolated from bone marrow (BM) by using c-kit microbeads. E10.5 aorta-gonad-mesonephros (AGM) ,E12.5、E14.5、E16.5 fetal liver (FL) and adult BM derived stromal cells (StroCs) were isolated and co-cultured with c-kit~+ HSPCs. The floating cells in co-culture system were sorted and counted by FACS. Gene expressions of Wnt and Notch pathway were detected by quantitative PCR and protein expressions by im-munostaining. Results Co-culturing HSPCs with AGM and FL-derived StroCs resulted in an expansion of c-kit~+ population from 0.4 × 10~5/well to (19.2 ±3.2) × 10~5/well and (26.8 ±5.4) × 10~5/well, respectively, being greater than that with BM-derived StroCs (P<0.05). The percentage of c-kit~+ cells detected in AGM-and BM- derived StroCs culture system was (75. 2 ±7. 1)% , (74.1 ±6.2)% respectively, being higher than FL- derived StroCs culture system (63.4 ±5.3)% (P<0.05). Wnt and Notch pathway genes expression varied at different phases of hematopoiesis. Wnt was highly expressed in AGM and FL derived StroCs, and, Notch did in AGM and BM derived StroCs. Conclusion Wnt and Notch pathway are important modulators in regulating time and spatial restricted hematopoiesis.