發(fā)布時間：2018-01-08 18:03

  本文關鍵詞：Wnt與Notch信號通路調(diào)控造血發(fā)育機制研究　出處：《華中科技大學》2010年碩士論文　論文類型：學位論文

  更多相關文章： 造血發(fā)育 C-kit~+造血祖細胞 Wnt通路 Notch通路

【摘要】：目的:研究在造血發(fā)育過程中不同階段不同部位,c-kit~+造血祖細胞中Wnt和Notch信號通路中各成分的時空表達變化。 方法:用免疫磁珠陽性分離法分離小鼠孕10.5天(E10.5)、E11.5 AGM區(qū),E12.5、E14.5、E16.5、E18胎肝(Fetal liver, FL)和成體骨髓(Bone marrow, BM)來源的c-kit陽性造血祖細胞,流式細胞術檢測其分選純度。實時定量PCR檢測以上時間點和部位分選的造血祖細胞中Wnt信號通路成員(wnt3a, wnt5a, wnt10b, Frizzled3, Frizzled4, Frizzled7, c-myc, ccnb1)和Notch信號通路成員(notch1, notch2, notch3, notch4, jagged1, hes1)的基因表達變化,免疫熒光染色法分別鑒定E10.5 AGM區(qū),E14.5 FL以及BM來源的c-kit~+細胞中WNT3a和NOTCH1蛋白的表達。將E10.5 AGM區(qū),E14.5 FL以及BM來源的c-kit~+細胞體外培養(yǎng)7天,檢測其增殖、分化以及集落形成能力。 結(jié)果:免疫磁珠陽性選擇法分選的c-kit~+細胞,純度可達90%以上。Wnt和Notch信號通路基因在各發(fā)育階段的c-kit~+造血祖細胞均有表達。其中,Wnt和Notch信號通路成員基因在AGM區(qū)來源的c-kit~+造血祖細胞中均高表達;胎肝來源c-kit~+造血祖細胞中Notch通路基因表達水平較低,明顯低于AGM區(qū)和骨髓來源c-kit~+造血祖細胞的表達;而骨髓來源的c-kit~+造血祖細胞中Wnt通路基因表達水平較低,明顯低于AGM區(qū)和FL來源c-kit~+造血祖細胞的表達(P 0.05)。免疫熒光染色顯示,在AGM區(qū),FL和BM來源的c-kit~+細胞中,WNT3a和NOTCH1蛋白均有不同程度的表達。E10.5 AGM區(qū)內(nèi)WNT3a和NOTCH1蛋白表達最高。與AGM區(qū)以及骨髓相比,FL來源的c-kit~+細胞NOTCH1蛋白的表達較低。與胎肝相比,骨髓來源的細胞WNT3a的表達明顯降低而NOTCH1蛋白的表達明顯升高(P 0.05)。體外培養(yǎng)7天后,AGM區(qū)和胎肝來源細胞具有較強的增殖能力,胎肝來源細胞易于分化,而骨髓來源細胞易于維持其未分化狀態(tài)。在體外集落形成能力方面,AGM區(qū)來源c-kit~+細胞具有較強的形成混合集落形成單位(CFU-Mix)的能力;而FL、BM來源c-kit~+細胞則能形成更多粒單系集落形成單位(CFU-GM)。 結(jié)論:造血發(fā)育過程中,不同發(fā)育階段的造血祖細胞的Wnt和Notch信號通路的活化程度不同,Wnt信號通路在E10.5 AGM區(qū)和E14.5胎肝中高度活化可能調(diào)控著造血祖細胞的增殖潛能。Notch信號通路在E10.5 AGM區(qū)和骨髓內(nèi)高度活化可能維持著造血祖細胞的未分化狀態(tài)。Wnt和Notch信號在造血發(fā)育過程中的調(diào)節(jié)具有時空限制性,它們相互協(xié)調(diào)精確調(diào)控著造血。
[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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329

【共引文獻】

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1 曾進龍;曾煉坤;王志超;張海良;毛平;;骨髓基質(zhì)細胞聯(lián)合細胞因子對臍血單個核細胞表面抗原表達的影響[J];中國組織工程研究與臨床康復;2011年36期

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1 宋麗娟;小鼠脂肪源間充質(zhì)干細胞在體外對造血功能影響的研究[D];新疆醫(yī)科大學;2013年

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本文編號：1398108