【摘要】：EPC是成熟血管內皮細胞的前體細胞，屬干細胞群體，和HSC來自同一祖先細胞。在胚胎，EPC參與胚胎發(fā)育的血管發(fā)生，出生后，與HSC共同存在于骨髓干細胞龕中，也存在于外周血、臍血中，具有缺血區(qū)定向歸巢并分化為成熟內皮細胞、促進受損內皮修復、血管新生等重要作用，不僅可應用于對于缺血性疾病的治療，對腫瘤的防治也有重要價值。 EPC最早是從外周血中分離CD34+細胞得到的，其表面標志被認定為CD34\CD133\KDR，，但是隨著進一步的研究發(fā)現(xiàn)EPCs是一組異質性的細胞群，它是由眾多處于不同分化階段的細胞組成，它的表面標記隨時間改變而變化。研究顯示，雖然不同的培養(yǎng)方法和細胞來源使EPC的表面標志不完全一致，但是目前體外培養(yǎng)的EPC有兩類，一類是梭型、增殖潛力有限、培養(yǎng)不超過8周的early EPC（EEPC），另一類是鵝卵石樣的、具有高增殖潛能、可以持續(xù)傳代培養(yǎng)的外向型生長內皮細胞late EPC (EOC)。研究顯示，EEPC和EOC都具有一定的血管形成和組織修復作用，但EEPC和EOC在血管的形成和受損組織修復的過程中具體發(fā)揮什么作用，與哪些信號通路和調控機制有關目前尚不清楚。 Notch信號途徑從胚胎發(fā)育到成年個體的多個系統(tǒng)中發(fā)揮重要作用，對不同組織中的細胞命運起著決定性作用。以往的研究提示Notch信號途徑對EPC的定植、遷移和歸巢有重要作用，我們前期的實驗也表明，小鼠肝臟部分切除后，EPC可以遷移至受損的肝臟組織參與肝組織的再生，但對EOC的影響未見報道。為了進一步分析Notch信號途徑對內皮干/祖細胞的調控機制，我們研究Notch信號途徑對不同分化階段的EPC的調控作用，由于轉錄因子RBP-J是Notch受體下游的關鍵效應分子，可以介導四種Notch受體在核內的轉錄激活作用，因此是整個Notch信號途徑調控的匯集點。我們通過建立RBP-J條件性剔除小鼠，擬表達了Notch在多種組織中的缺失，研究結果顯示Notch信號途徑在維持血管平衡和組織修復方面有重要的作用。 主要研究成果如下： 1．通過體外貼壁擴增培養(yǎng)，從小鼠骨髓細胞中成功培養(yǎng)出EEPC和EOC，表達CD34+/CD133+/VEGFR2+的EEPC數(shù)目從最初的0.08%能夠增長至50%以上； 2．我們發(fā)現(xiàn)，在三維管腔形成實驗中，EEPC不能形成管腔樣結構，而EOC能夠形成管腔樣結構并且受到Notch-RBP-J信號通路的調控；阻斷Notch-RBP-J信號通路后，EEPC的增殖，遷移，CXCR4的表達均呈下降趨勢而EOC的增殖、遷移以及CXCR4的表達呈上升趨勢。提示Notch信號通路對EEPC和EOC的調控不相同； 3．通過RBP-J條件性基因剔除小鼠模型，我們進一步觀察到，小鼠肝大部切除術后（PHx），通過Notch信號通路的調控EEPC可以迅速募集到受損肝臟，促進肝臟血管的重建、肝細胞的增殖以及肝功能的恢復，而EOC并沒有有效促進肝臟的再生和肝細胞的增殖，提示Notch-RBP-J信號通路在EEPC和EOC參與的肝再生進程中的調控作用不同，并且發(fā)現(xiàn)EEPC和EOC在肝再生過程中發(fā)揮著不同的作用。 綜上所述，我們通過RBP-J剔除小鼠，觀察了Notch-RBP-J信號對于EEPC和EOC參與的肝再生進程的調控作用。我們的結果表明， Notch信號參與調控EEPC和EOC對肝細胞的再生，肝臟的修復，以及肝功能的恢復，并且EEPC和EOC的增殖、分化和遷移受到Notch-RBP-J信號途徑的調控；EEPC和EOC在肝再生進程中發(fā)揮著不同的作用。這些研究為進一步了解內皮干/祖細胞的功能，深入分析Notch對內皮干/祖細胞的調控機制奠定了基礎，
[Abstract]:The EPC is the precursor cell of mature vascular endothelial cells, the stem cell population, and the HSC from the same ancestor cell. In the embryo, the EPC participates in the angiogenesis of the development of the embryo, and is co-existing with the HSC in the bone marrow stem cell niche after birth, and is also present in the peripheral blood and the umbilicus blood, has the important effects of directional homing and differentiation of the ischemic region into the mature endothelial cells, promoting the repair of the damaged endothelium, the angiogenesis and the like, Not only can be applied to the treatment of ischemic diseases, but also has important value for preventing and treating the tumor. EPC was the first to separate CD34 + cells from peripheral blood and its surface markers were identified as CD34CD133KDR, but with further study, EPCs were a heterogeneous group of cells, consisting of numerous cells at different stages of differentiation, whose surface markers varied with time The results show that, although the different methods of culture and the source of the cell make the surface marker of the EPC not exactly the same, there are two types of EPC in vitro, one is the shuttle type, the proliferation potential is limited, the early EPC (EEPC) with no more than 8 weeks is cultured, and the other is a cobblestone, which has high proliferation potential. an extraversion growth endothelial cell (EOC) EPC (EOC) that can be continuously subcultured ). The study shows that both the EEPC and the EOC have certain vascular formation and tissue repair, but the EPCs and EOC play a specific role in the formation of the blood vessels and the repair of the damaged tissue, and which signal pathways and regulatory mechanisms are still unclear. The Notch signaling pathway plays an important role in a number of systems from the development of the embryo to the adult, and plays a decisive role in the fate of the cells in different tissues The previous study suggests that the Notch signaling pathway plays an important role in the field planting, migration and homing of the EPC, and the earlier experiments have shown that the EPC can be migrated to the damaged liver tissue to participate in the regeneration of the liver tissue after the partial hepatectomy of the mouse, but the effect on the EOC is not In order to further analyze the regulation mechanism of Notch signaling pathway on endothelial stem/ progenitor cells, we study the regulation of Notch signaling pathway on the EPC of different stages of differentiation, because the transcription factor, RBP-J, is the key effect downstream of the Notch receptor. The transcription activation of the four Notch receptors in the nucleus can be mediated by the molecule, so it is regulated by the whole Notch signaling pathway. The results show that the Notch signaling pathway plays an important role in the maintenance of vascular balance and tissue repair by establishing a RBP-J conditional knockout mouse. main research The results were as follows:1. The number of EEPCs expressing CD34 +/ CD133 +/ VEGFR2 + from the bone marrow cells of mice was increased from the original 0.08% by the in vitro adherent amplification culture. up to 50% or more;2. We found that in In the three-dimensional lumen formation experiment, the EPCs can not form a tube-cavity-like structure, and the EOC can form a tube-cavity-like structure and is controlled by the Notch-RBP-J signal path; after blocking the Notch-RBP-J signal path, the proliferation and migration of the EEPC, the expression of the CXCR4 are all down, and the proliferation, the migration and the CXCR of the EOC The expression of the 4 is on the rise. The Notch signal path is prompted for the EEPC and The control of EOC was not the same;3. The mouse model was removed by the RBP-J conditional gene. We further observed that after the partial hepatectomy (PHx), the EEPC through the Notch signaling pathway could be quickly raised to the damaged liver. It was found that the regulation of the Notch-RBP-J signaling pathway in the liver regeneration process involved in the participation of EEPC and EOC was different, and EEPC and EOC were found to be in the liver regeneration. In conclusion, we removed the mice by RBP-J and observed the Notch-RBP-J signal for EEPC and EOC The results showed that the Notch signal was involved in the control of the regeneration of the liver cells, the repair of the liver, and the recovery of the liver function, and the proliferation, differentiation and migration of the EEPC and the EOC were regulated by the Notch-RBP-J signaling pathway, and the EEPC and EOC were in the liver. Different roles have been played in the regeneration process. These studies provide a further understanding of the function of endothelial stem/ progenitor cells, and in-depth analysis of Notch's effect on the endothelium/ progenitor cells
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R363

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