Wnt与Notch信号通路调控造血发育机制研究

发布时间：2018-01-08 18:03

本文关键词：Wnt与Notch信号通路调控造血发育机制研究　出处：《华中科技大学》2010年硕士论文　论文类型：学位论文

【摘要】：目的:研究在造血发育过程中不同阶段不同部位,c-kit~+造血祖细胞中Wnt和Notch信号通路中各成分的时空表达变化。方法:用免疫磁珠阳性分离法分离小鼠孕10.5天(E10.5)、E11.5 AGM区,E12.5、E14.5、E16.5、E18胎肝(Fetal liver, FL)和成体骨髓(Bone marrow, BM)来源的c-kit阳性造血祖细胞,流式细胞术检测其分选纯度。实时定量PCR检测以上时间点和部位分选的造血祖细胞中Wnt信号通路成员(wnt3a, wnt5a, wnt10b, Frizzled3, Frizzled4, Frizzled7, c-myc, ccnb1)和Notch信号通路成员(notch1, notch2, notch3, notch4, jagged1, hes1)的基因表达变化,免疫荧光染色法分别鉴定E10.5 AGM区,E14.5 FL以及BM来源的c-kit~+细胞中WNT3a和NOTCH1蛋白的表达。将E10.5 AGM区,E14.5 FL以及BM来源的c-kit~+细胞体外培养7天,检测其增殖、分化以及集落形成能力。结果:免疫磁珠阳性选择法分选的c-kit~+细胞,纯度可达90%以上。Wnt和Notch信号通路基因在各发育阶段的c-kit~+造血祖细胞均有表达。其中,Wnt和Notch信号通路成员基因在AGM区来源的c-kit~+造血祖细胞中均高表达;胎肝来源c-kit~+造血祖细胞中Notch通路基因表达水平较低,明显低于AGM区和骨髓来源c-kit~+造血祖细胞的表达;而骨髓来源的c-kit~+造血祖细胞中Wnt通路基因表达水平较低,明显低于AGM区和FL来源c-kit~+造血祖细胞的表达(P 0.05)。免疫荧光染色显示,在AGM区,FL和BM来源的c-kit~+细胞中,WNT3a和NOTCH1蛋白均有不同程度的表达。E10.5 AGM区内WNT3a和NOTCH1蛋白表达最高。与AGM区以及骨髓相比,FL来源的c-kit~+细胞NOTCH1蛋白的表达较低。与胎肝相比,骨髓来源的细胞WNT3a的表达明显降低而NOTCH1蛋白的表达明显升高(P 0.05)。体外培养7天后,AGM区和胎肝来源细胞具有较强的增殖能力,胎肝来源细胞易于分化,而骨髓来源细胞易于维持其未分化状态。在体外集落形成能力方面,AGM区来源c-kit~+细胞具有较强的形成混合集落形成单位(CFU-Mix)的能力;而FL、BM来源c-kit~+细胞则能形成更多粒单系集落形成单位(CFU-GM)。结论:造血发育过程中,不同发育阶段的造血祖细胞的Wnt和Notch信号通路的活化程度不同,Wnt信号通路在E10.5 AGM区和E14.5胎肝中高度活化可能调控着造血祖细胞的增殖潜能。Notch信号通路在E10.5 AGM区和骨髓内高度活化可能维持着造血祖细胞的未分化状态。Wnt和Notch信号在造血发育过程中的调节具有时空限制性,它们相互协调精确调控着造血。
[Abstract]:Aim: to study the temporal and spatial expression of Wnt and Notch signaling pathway in c-kit-hematopoietic progenitor cells at different stages of hematopoietic development. Methods: E12.5E14.5E16.5 was isolated from the E11.5 AGM region of E10.5 AGM by immunomagnetic bead positive method. E18 fetal liver Fetal liver (FLL) and adult bone marrow bone marrow-derived c-kit positive hematopoietic progenitor cells. Flow cytometry (FCM) was used to detect its sorting purity. Real-time quantitative PCR was used to detect the members of Wnt signaling pathway such as wnt3a, wnt5a, wnt10b in hematopoietic progenitor cells. Frizzled3, Frizzled4, Frizzled7, c-myc, ccnb1) and a member of the Notch signaling pathway, notch1. The gene expression changes of notch2, notch3, notch4, jagged1, Hes1) were detected by immunofluorescence staining. The E10.5 AGM region was identified by immunofluorescence staining. Expression of WNT3a and NOTCH1 protein in E14.5FL and BM-derived c-kit~ cells. E10.5 AGM region was obtained. E14.5FL and BM-derived c-kit~ cells were cultured for 7 days, and their proliferation, differentiation and colony forming ability were detected. Results: c-kit~ cells were separated by immunomagnetic bead positive selection method. Wnt and Notch signaling pathway genes were expressed in c-kit~ hematopoietic progenitor cells at all stages of development. Wnt and Notch signaling pathway genes were highly expressed in c-kit~ hematopoietic progenitor cells derived from AGM region. The expression level of Notch pathway gene in fetal liver derived c-kit~ hematopoietic progenitor cells was lower than that in AGM region and bone marrow-derived c-kit~ hematopoietic progenitor cells. However, the expression level of Wnt pathway gene in bone marrow-derived c-kit~ hematopoietic progenitor cells was lower. The expression of c-kit~ hematopoietic progenitor cells in AGM and FL was significantly lower than that in FL-derived hematopoietic progenitor cells (P 0.05). Immunofluorescence staining showed that it was in the AGM region. C-kit~ cells from FL and BM. The expression of WNT3a and NOTCH1 in the region of E10.5 AGM was the highest in WNT3a and NOTCH1, compared with AGM and bone marrow. The expression of NOTCH1 protein in c-kit~ cells derived from FL was lower than that in fetal liver. The expression of WNT3a in bone marrow-derived cells was significantly decreased, while the expression of NOTCH1 protein was significantly increased (P 0.05). AGM region and fetal liver derived cells have strong proliferative ability, fetal liver derived cells are easy to differentiate, while bone marrow derived cells are easy to maintain their undifferentiated state. The c-kit~ cells derived from the AGM region had a strong ability to form CFU-Mix. The c-kit~ cells derived from FLN BM could form more monolith colony forming units (CFU-GMN). Conclusion: during hematopoietic development, the activation degree of Wnt and Notch signaling pathway of hematopoietic progenitor cells is different in different stages of hematopoietic development. High activation of Wnt signaling pathway in E10.5 AGM region and E14.5 fetal liver may regulate proliferation potential of hematopoietic progenitor cells. Notch signaling pathway in E10.5. The high activation of AGM region and bone marrow may maintain the undifferentiated state of hematopoietic progenitor cells. Wnt and Notch signals are spatiotemporal restricted during hematopoietic development. They coordinate and precisely regulate hematopoiesis.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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