- 學位論文
Wnt3a對心臟Nkx2.5+ cardiomyoblast、纖維母細胞、心肌細胞不同之調控效果
Different Modulatory Effect of Wnt3a in Nkx2.5+ Cardiomyoblast, Cardiac Fibroblast and Cardiomyocyte
若您是本文的作者，可授權文章由華藝線上圖書館中協助推廣。
摘要
簡介：Cardiac stem/progenitor cell被發現存在於在心臟之中，這些細胞具有自我修復之能力。Wnt signaling在胚胎的心臟發育及許多心臟疾病有著重要地位，這些疾病包括了心肌梗塞、心衰竭、以及心臟肥大。但Wnt對於心臟內各種細胞產生的各種不同作用，至今仍不清楚。目的：本篇論文將釐清Wnt3a在Nkx2.5+ cardiomyoblasts之self-renewal、心臟纖維母細胞的生長及transdifferentiation、心肌細胞的存活中，所扮演的腳色。 材料與方法：本篇論文利用具有Nkx2.5+ enhancer-GFP的基因轉殖鼠，使心臟中高度表現Nkx2.5之幹細胞表達GFP。利用酵素分解及flow cytometry將心室中的Nkx2.5-GFP+ cardiomyoblasts分離出來；也以酵素分解方式游離出纖維母細胞及成鼠心肌細胞。以CyQuant asssay或BrdU-pulse-labeling assay來了解Wnt對cell proliferation之影響，用免疫螢光染色來檢測細胞的分化情形，qPCR分析細胞轉錄特徵。 實驗結果：Wnt3a會產生劑量依存性地抑制Nkx2.5+ cardiomyoblast的生長，且這樣的效果可被一些Wnt抑制劑減少，如DKK1 (Wnt抑制劑)，KY02111 (canonical Wnt抑制劑)、Y27632 (ROCK抑制劑)。而在心臟纖維母細胞中，Wnt3a可以抑制這些細胞進入cell cycle，但這樣的效應卻不能被KY02111所抵消。CHIR (a GSK3β inhibitor)會減少纖維母細胞中表現α-smooth muscle actin (α-SMA)的細胞，Wnt3a同樣可減少表現α-smooth muscle actin (α-SMA)的細胞量，且這效果能被KY02111所減少。此外，Wnt3a並不會影響體外培養成鼠心肌細胞的存活率。若將心肌細胞與Nkx2.5+ cardiomyoblast共同培養至五天，Nkx2.5+ cardiomyoblast可增加心肌細胞的存活，而Wnt3a會抑制Nkx2.5+ cardiomyoblast提供的支持心肌細胞存活效果。由qPCR檢驗Wnt receptor subtypes (Fzd)的轉錄表現量發現Fzd2及Fzd1(Wnt3a作用之receptor)在Nkx2.5+ cardiomyoblast及纖維母細胞中高度表現，且Fzd2在Nkx2.5+ cardiomyoblast中表現量遠高於纖維母細胞。 總結：Wnt3a可經由Wnt canonical (KY02111可抑制)及non-canonical pathway (Y27632可抑制)來減少Nkx2.5+ cardiomyoblast之生長。此情形與Wnt3a對纖維母細胞生長的抑制狀況不同，KY02111無法反轉Wnt3a的抑制作用。然而，Wnt3a抑制纖維母細胞轉型分化成myofibroblast的效應，卻可被KY02111抑制。另外，Wnt3a不會直接影響成鼠心肌細胞的存活或誘發細胞肥大。但在成鼠心肌細胞與Nkx2.5+ cardiomyoblast共同培養條件下，Wnt3a會抑制Nkx2.5+ cardiomyoblast產生的旁泌作用，減弱支持心肌細胞存活的效果。關於Wnt調控細胞生長、分化的詳細機制，以及如何影響Nkx2.5+ cardiomyoblast旁泌作用，仍需進一步研究探討。
並列摘要
Introduction: Cardiac stem/progenitor cells were found resident in the heart and possess the potential for the self-repair. Wnt signaling plays a pivotal role in embryonic cardiogenesis and was found to be activated under diseases, including myocardial infarction, heart failure, and cardiac hypertrophy. It is still unclear in the different functioning of Wnts in cardiac cells. Aim: The present study aimed to clarify the different functional role of Wnt3a in regulating the self-renewal of Nkx2.5+ cardiomyoblasts, the growth and the transdifferentiation of cardiac fibroblast, and the survival of cardiomyocytes. Material and Method: Nkx2.5-GFP+ cardiomyoblasts were isolated by enzymatic method from the ventricle of a reporter mice expressing GFP driven by an Nkx2.5-enhancer, and were sorted by flow cytometry. Enzyme digestion was also used to isolate fibroblasts and adult cardiomyocytes. CyQuant asssay or BrdU-pulse-labeling assay was performed to examine the effect of Wnts on cell proliferation. The differentiation pattern was evaluated by immunostaining and transcriptional profile by qPCR. Results: Wnt3a, but not Wnt4, could dose-dependently inhibit the self-renewal of Nkx2.5+ cardiomyoblast, and the effect could be attenuated in the presence of Wnt inhibitors, including KY02111, DKK1, and a ROCK inhibitor (Y27632). In cardiac fibroblasts, Wnt3a could inhibit fibroblast entering cell cycle revealed by BrdU-pulse-labeling assay, which could not be blocked by KY02111. Furthermore, Wnt3a could decrease the cells expressing α-smooth muscle actin (α-SMA) and the effect could be attenuated by KY02111. CHIR (a GSK3β inhibitor) could also reduce α-SMA+ cells of cultured fibroblast. In cultured adult cardiomyocytes, Wnt3a did not alter myocyte survival in vitro. Co-culture of myocytes with Nkx2.5+ cardiomyoblast could enhance myocyte survival after culture for 5days, which could be attenuated by Wnt3a. The transcriptional profile of Wnt-receptor subtypes (Fzds) was analyzed by qPCR. High transcriptional levels of Fzd2 and Fzd1, the binding receptors of Wnt3a, were found in both Nkx2.5+ cardiomyoblast and cardiac fibroblast, and Nkx2.5+ cardiomyoblast expressed higher Fzd2 than fibroblast. Conclusion: Wnt3a could inhibit the proliferation of Nkx2.5+ cardiomyoblast via both canonical Wnt-signaling (blocked by KY02111) and non-canonical pathway (blocked by Y27632). It was different from the inhibitory effect of Wnt3a in cardiac fibroblast proliferation that was insensitive to KY02111. KY02111 could reverse Wnt3a-induced reduction of myofibroblast transdifferentiation. Wnt3a did not alter adult cardiomyocyte survival or induce cell hypertrophy after in-vitro 3-day-culture, but attenuated the paracrine benefit of Nkx2.5+ cardiomyoblast to enhance myocyte survival after 5-day-culture under co-culture condition. It needs further study to clarify the detailed Wnt mechanisms corresponding to the regulation in cell proliferation, differentiation and the synthesis of paracrine factors in Nkx2.5+ cardiomyoblast. Key words：cardiac progenitor cell, Nkx2.5, Wnt3a, Wnt signaling pathway, cardiac fibroblasts, cardiomyocytes
參考文獻
Aisagbonhi, O., M. Rai, et al. (2011). "Experimental myocardial infarction triggers canonical Wnt signaling and endothelial-to-mesenchymal transition." Dis Model Mech 4(4): 469-483.
Beltrami, A. P., L. Barlucchi, et al. (2003). "Adult cardiac stem cells are multipotent and support myocardial regeneration." Cell 114(6): 763-776.
Bergmann, M. W. (2010). "WNT signaling in adult cardiac hypertrophy and remodeling: lessons learned from cardiac development." Circ Res 107(10): 1198-1208.
Blankesteijn, W. M., Y. P. Essers-Janssen, et al. (1996). "Increased expression of a homologue of drosophila tissue polarity gene "frizzled" in left ventricular hypertrophy in the rat, as identified by subtractive hybridization." J Mol Cell Cardiol 28(5): 1187-1191.
Caricasole, A., T. Ferraro, et al. (2003). "Functional characterization of WNT7A signaling in PC12 cells: interaction with A FZD5 x LRP6 receptor complex and modulation by Dickkopf proteins." J Biol Chem 278(39): 37024-37031.