2013-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/659278摘要：SCIPIO (Stem Cell Infusion in Patients with Ischemic cardiOmyopathy)第一期臨床試驗成功地分離人類成體心臟幹細胞，於體外培養擴增後，再經由冠狀動脈輸注回病人病灶區，追蹤發現一年後梗塞面積顯著減少，並有效改善左室收縮功能。然而，促進成體心臟幹細胞生長機制仍不清楚，需要探索有效擴增成體心臟幹細胞的方法，以利心臟修復和再生。 表現 Nkx2.5的心臟幹細胞（Nkx2.5+ CSCs）為哺乳動物胚胎發育過程中的心臟血管細胞前驅。藉由 Nkx2.5 enhancer-eGFP轉基因報告小鼠標記 Nkx2.5+ CSCs表現 GFP，可在出生後和成體小鼠心臟中發現存留的 Nkx2.5+ CSCs。我們前期的研究發現 A83-01（TGFβRI 抑制劑）可經由 MEK 相關途徑，刺激 Nkx2.5+ CSCs生長，同時發現 survivin, VEGFD, FGFR2, -catenin之轉錄表現增加，而 Fzd6 (frizzled 6 Wnt受體)與 TGF-β1之表現減少。我們已證實Wnt3a會抑制 Nkx2.5+ CSCs生長。然而，調控 Nkx2.5+ CSCs增殖和分化的詳細訊息網路需要進一步研究釐清，以找出理想的藥理標的及作用藥物。 基於此，本三年期研究計畫的目標在(1) 第一年：探究抑制 TGF-βRI 後如何抑制 Fzd6並增加-catenin表現，釐清典型及非典型Wnt訊息在Nkx2.5+ CSCs的功能及調控路徑，(2) 第二年：釐清-catenin在 Nkx2.5+ CSCs的功能角色，瞭解其調控 survivin, VEGFD, FGFR2表現與促進生長的關係，並釐清其旁泌功效。(3) 第三年：找出不會影響 TGF-β1抗發炎作用，又能促進 CSCs增生之藥理標的及調控小分子，於心肌梗塞小鼠測試其促進修復受傷心肌潛力。並可將CSCs與促進CSCs增生小分子以微珠支架包裹殖入心肌梗塞疤痕區或代償失調性心衰竭心臟中，評估修復心肌和再生組織工程轉譯潛力。 此分子與藥理機制研究，能提供調控成體心臟幹細胞生長及分化之新見解。<br> Abstract: The encouraging phase 1 report of SCIPIO (Stem Cell Infusion in Patients with Ischemic cardiOmyopathy) clinical trial showed that adult human cardiac resident stem/progenitor cells (CSCs) can be isolated and expanded ex vivo, and demonstrated that intracoronary infusion of autologous CSCs is effective in improving LV systolic function and reducing infarct size in patients with heart failure after myocardial infarction. However, the self-renewal mechanism of CSC is still unclear and it is crucial to discover the effective methods to repopulate the resident CSCs for cardiac repair and regeneration. A cardiac-specific Nkx2.5+ cell population is a common precursor for cardiovascular lineages in the developing mammalian embryo heart. Using the Nkx2.5 enhancer-eGFP transgenic reporter mice to label cardiac Nkx2.5+ cells with an embryonic phenotype, we have previously found that a population of the resident Nkx2.5+ CSCs can be identified in the hearts of postnatal and adult mice and possess the potential to differentiate into cardiomyocytes or smooth muscle cells. Our previous study demonstrated that TGFRI inhibition can enhance resident Nkx2.5+ CSC proliferation in vitro and in vivo via MEK-dependent manner in association with the change of transcriptional profile including the up-regulation of survivin, VEGFD, FGFR2, and -catenin as well as the down-regulation of frizzled 6 Wnt receptor (fzd6) and TGF-1. Wnt3a could inhibit Nkx2.5+ CSC growth and induced cells expressing smMHC. However, it remains unclear in the underlying mechanisms, and needs further investigation to clarify the signaling networks in CSCs for searching the pharmacological targets and modulators for CSC expansion. Based on our previous study, the goals of the present three-year project are to clarify the mechanisms including (1) how TGFRI inhibition could reduce fzd6 and increase -catenin, and the functional role of canonical and non-canonical Wnt-signaling in Nkx2.5+ CSC (in the first year), (2) the role of -catenin in regulating the expression of survivin, VEGFD and FGFR2 as well as the cause-effect relationship to MEK-dependent CSC proliferation (in the second year). The paracrine effect of Nkx2.5+ CSC will be examined as well. Therefore, we can identify the ideal pharmacological targets and modulators that can repopulate CSC without interfering with the anti-inflammatory action of TGF-, and assess drug efficacy in facilitating cardiac repair in post-MI mice (in the third year). Furthermore, we will optimize the condition of CSC number and drug dosing in the microenvironment of alginate microsphere for intra-myocardial injection of the microsphere into the scar region of infarcted hearts or the decompensated heart to assess the regenerative efficacy (in the third year). The arrhythmogenicity and functional properties of the engrafted cells will be examined as well. The three-year project can offer new insights into the molecular and pharmacological mechanisms in regulating self-renewal and differentiation of adult resident CSCs.心臟幹細胞/前驅細胞Nkx2.5心肌梗塞心臟再塑心臟再生Cardiac stem/progenitor cellNkx2.5myocardial infarctioncardiac remodelingcardiac regeneration.