- 學位論文
利用來自左心室緻密化不全心肌病變病人之誘導式多能幹細胞分化得到的心肌細胞做為疾病模擬和探索治療藥物
Using human induced pluripotent stem cell derived cardiomyocytes for disease modelling of left ventricular non-compaction cardiomyopathy and therapeutic drug discovery
摘要
擴張型心肌病會導致左心室之擴大與心臟收縮力之減弱。造成擴張型心肌病的各式病因限制了我們對於長期發病機制的了解。有個獨特的家族性擴張型心肌病合併顯著的左心室心肌緻密化不全家族，被鑑別其肌節基因上有兩個突變(TNNT2:R151W and MYPN:S1296T)。此外，帶有此突變的女兒的擴張型心肌病的進程比有同樣突變的爸爸更為快速。對於等待移植的小兒患者來說，可用的器官捐贈者的缺乏，導致死亡率居高不下。因此，建立個人化的基因特異性治療是相當重要的。本研究旨在利用擴張型心肌患者的誘導性多功能幹細胞所分化的心肌細胞，探討相關機制及治療藥物。 實驗發現此家族性擴張型心肌病合併顯著的左心室心肌緻密化不全家族患者的誘導性多功能幹細胞所分化的心肌細胞可成功的重現疾病的特徵，在分化大概五十天後，會有細胞收縮減弱及異常的胞內鈣離子處理等等特性。實驗亦發現在此類擴張型心肌病患者的誘導性多功能幹細胞所分化的心肌細胞中，有變異的TNNT2核轉移的現象，與PDE3A的表達量增加與PDE9A的表達量減少有關聯。此外，在分化約50天的此類擴張型心肌病患者的誘導性多功能幹細胞所分化的心肌細胞中，cilostazol (PDE3的抑制劑)可以顯著的增加細胞收縮強度。在分化後第30天給予statins類藥物治療(simvastatin)，可顯著改善心肌細胞之收縮功能障礙。在之後的研究中，我們會繼續確認statin的給予與PDEs表現量的改變。 在表達變異的TNNT2的HL-1細胞中，我們發現他們對於β-AR有較差的反應，這與PDE9A的表達量下降有關聯，且可以被statin的治療改善。但在HL-1的細胞中，statin的治療無法改善變異的TNNT2轉移到細胞核的現象，但可以顯著回復PDE9A的表達量，改善細胞的大小與β-AR刺激的收縮反應。 總結來說，此研究建立了家族性擴張型心肌病合併顯著的左心室心肌緻密化不全家族患者的誘導性多功能幹細胞所分化的心肌細胞之實驗平台，可用於做機轉之研究與治療藥物的開發。Simvastatin被認為在體外的實驗中有可能改善心肌功能不全的原因與在表現變異的R151W troponin T的心肌細胞中可使PDE9A的表現量正常化有關。這需要全面的遺傳分析與功能測試以確認是否在其他的基因上有變異，包含notch與TBX10，有助於了解左心室心肌緻密化不全的病理機轉與statins類藥物可改善心肌功能不全的機轉，以達到個別化治療與精確的醫療之目標。
並列摘要
Dilated cardiomyopathy (DCM) causes left ventricular dilation and systolic failure. The heterogeneous etiologies underlying DCM limited our understanding of the long-term pathogenesis of DCM. A unique familial DCM family with significant left ventricular noncompaction (LVNC) was identified with two mutations in the sarcomeric genes (TNNT2:R151W and MYPN:S1296T). Furthermore, LVNC progression in the daughter carrying the mutations was faster than that in the LVNC father. The lack of available donors results in significant mortality for pediatric patients awaiting transplantation. Thus, it is appealing to establish the individualized gene-specific therapy. Using cardiomyocytes derived from LVNC-hiPSC-CM, the present study aimed to investigate the underlying mechanism and to identify the therapeutic drugs. Our study found that hiPSC-CM derived from these LVNC families could successfully recapitulate the disease phenotype with the decreased cell shortening and the abnormal intracellular calcium handling property around day 50 after differentiation. It was also found the nuclear translocation of the mutant TNNT2 in LVNC-derived hiPSC-CM in association with the increase of PDE3A and the decrease of PDE9A expressions. Furthermore, cilostazol (a PDE3 inhibitor) could significantly increase cell shortening in LVNC-iPSC-CM on day 70. The contractile dysfunction could be significantly reversed with the treatment of statins (simvastatin) since day 30 after differentiation. It will be further confirmed the alteration of PDEs expressions in LVNC-hiPSC-CM treated with statins. In HL-1 cell expressing mutant TNNT2 (TNNT(R151W)-HL-1), it was found a less β-AR response in association with the decrease of PDE9A, which could be reversed in the presence of simvastatin. Simvastatin did not inhibit the nuclear translocation of mutant TNNT2 in TNNT(R151W)-HL-1, but could significantly recover PDE9A expression, cell size, and β-AR-stimulated positive inotropic response. In conclusion, this study established a LVNC-hiPSC-CM platform to characterize the pathogenesis for mechanic study and the development of therapeutic drugs. Simvastatin was identified with the benefit to potentially ameliorate cardiac dysfunction in vitro in association with the normalization of PDE9A expression in the myocytes expressing mutant R151W troponin T. It needs comprehensive genetic analysis with combination of the functional study to clarify the mutations in other genes, including notch and TBX10, contribute to the pathogenesis of this LVNC family and the mechanistic link to the action of statins in the improvement of cardiac function for the goals of the individualized therapy and precision medicine.