人體周邊血促進血管生成之類內皮前驅細胞的分離培養及鑑定
Isolation and characterization of human peripheral blood derived endothelial progenitor-like cells for neo-vasculogenesis
中文摘要
內皮前驅細胞能夠修復受傷的血管，促進血管的再生，本實驗分離健康成人之週 邊血單核細胞，利用特殊的培養方法，促使貼附性單核細胞分化為類內皮前驅細 胞，並鑑定其特徵及功能。自體外培養七天後得到的類內皮前驅細胞，具有攝取 DiI-Ac LDL 以及結合 ULEX-1 的功能，此特徵與前人所報導的內皮前驅細胞相 像，並且藉著反轉錄聚合?連鎖反應以及免疫螢光染色，此細胞也表現內皮細胞 之特有基因：VE-cadherin、vWF、eNOS，以及內皮細胞之特有蛋白：vWF。在 細胞的功能性分析方面，得知類內皮前驅細胞在體外的細胞間質萃取物
(Matrigel) 上可以促使類似血管的生成；藉由流式細胞儀偵測培養天數不同的細 胞表面分子表現：血源性細胞標的：CD105 會隨著培養天數大幅降低，另外，
早期內皮細胞的內皮血管生長因子接受器 1 & 2 (Flt-1 & KDR) 隨著天數表現則 逐漸升高，顯示血源性單核細胞轉分化 (trans-differentiation) 的趨勢。此外，在 形成類內皮前驅細胞的過程，可以偵測到代表早期細胞的標記基因：Oct-4、
Rex-1、Sox-2 的表現逐漸增高，並且細胞端粒?活性也增強，顯示成人細胞在經 由培養之後會表現早期細胞的特性。進一步了解內皮前驅細胞分化過程中分泌了 哪些激素，收集其第一天至第四天以及第四天至第七天，類內皮前驅細胞形成過 程的培養液，以人類細胞激素蛋白表達微陣列之比對分析，可以得知養成類內皮 前驅細胞第四天至第七天，細胞會大量表現：Hemangiogenic
factors/Pro-angiogenic factors
(b-FGF、G-CSF and GM-CSF)，Chemokines/Pro-inflammatory
factors/Pro-angiogenic factors (GRO/CXCL-2、IL-8/CXCL-8、 I-309/CCL-1、
I-TAC/CXCL-11、MCP-1/CCL-2、RANTES/CCL-5) 以及
Cytokine/Pro-inflammatory factors (IL-13) 這些細胞激素，可能經由 paracrine 或 autocrine 的機制調控其本身的分化，促使血管的生成及持久性和穩定性，或者扮 演其他生理性功能。
經由以上的實驗結果證明在體外 (in vitro) 的情況下可以使成人週邊血中的 血源系細胞轉分化或去分化成為類內皮前驅細胞，並且可能經由細胞激素的刺激 促進血管的再生，進一步將利用動物實驗證實。
英文摘要
This study describes the generation and characterization of human adult peripheral blood mononuclear-derived endothelial progenitor-like cells (EPLCs) as a resource may potentially useful for neo-vasculogenesis.
Peripheral blood mononuclear cell derived EPLCs are characterized by their phonotypical expression of CD34–, CD105+, CD14++, CD31++, CD45+++, and VEGF receptors positive (Flt-1++ and KDR++). These cells up-taking AC-LDL, binding ULEX-1 functions and exhibit tube formation in the Matrigel culture system in resemble to the CD34+, KDR++, AC133+ endothelial progenitor cells (EPCs).
EPLCs express endothelial-specific genes such as VE-cadherin, vWF, and eNOS as functioning endothelial cells (ECs). During the course of EPLCs formation culture, increased early genes (Oct-4, Rex-1, and Sox-2) expression and enhanced telomerase activity were detected as their positive cell surface marker CD105 was switched off and at mean time turned on the angiogenic receptors Flt-1, and KDR. In contrast to the previously finding of angiogenic factors G-CSF, VEGF, HGF secreted by the EPCs18, the current study reveals that EPLCs secrete additional hemangiogenic factors/pro-angiogenic factors (b-FGF、GM-CSF), chemokines/pro-inflammatory factors/pro-angiogenic factors (GRO/CXCL-2、IL-8/CXCL-8、 I-309/CCL-1、
I-TAC/CXCL-11、MCP-1/CCL-2、RANTES/CCL-5), and cytokine/pro-inflammatory factors (IL-13).
Taken together, the present study demonstrates that EPLCs can be derived from transforming human adult hematopoietic mononuclear cells into functioning
endothelial progenitor like cells phenotype via the process of trans-dedifferentiation in vitro that may benefit for the vascular wound healing and regeneration in vivo.
Animal model studies of the EPLCs are undergoing.
