以病患誘導性多潛能幹細胞研究體顯性多囊腎心臟血管併發症之細胞表現及疾病機轉

Cellular phenotypes and disease mechanisms of autosomal dominant polycystic disease patients’ cardiovascular complication using patient induced pluripotent stem cells

李佳蓉(Jia-Jung Lee)

指導教授 ： 謝清河

高雄醫學大學/醫學院/轉譯醫學博士學位學程/博士(2019年)

摘要

台灣的末期腎病患者發生率及盛行率世界最高，體顯性多囊腎是腎衰竭的第四大病因，也是盛行率最高的單一基因致病的腎臟疾患。罹患體顯性多囊腎，除了雙側腎水囊及逐漸惡化的腎衰竭之外，患者常合併有其他器官系統的病症，其中，心臟血管併發症發生率高並且是多囊腎患者最常見的死因。此關聯性是間接的心腎症候群，亦或是源自於突變基因在心臟血管細胞的直接影響，仍屬未知，目前臨床上仍欠缺體顯性多囊腎及其併發症的有效治療。
近年文獻發現，體顯性多囊腎的致病基因 PKD1 及 PKD2 及其所製造的蛋白質 polycystin1和 polycystin2具有對動物心肌細胞的影響。本研究假設，體顯性多囊腎的致病基因直接有心肌細胞的影響，進而導致臨床上病患的心臟血管併發症。基於人類細胞和動物細胞在酵素系統及離子通道上的差異，動物研究模式也難以區分心臟腎臟之間直接或間接的作用，本研究運用病患細胞產製的誘導性多潛能幹細胞，首先將病患的誘導性多潛能幹細胞細胞分化成類心肌細胞，這些細胞表現心肌蛋白，並且會如同心臟一樣規律的跳動。以電生理的實驗方法分析發現，由多囊腎病患幹細胞所衍生的心肌細胞，其跳動型態及對藥物的反應和原病患幾乎相同。進一步的研究更顯示，體顯性多囊腎病患幹細胞所衍生的心肌細胞具有心律不整的傾向，此發現和臨床上體顯性多囊腎病患的高心臟血管併發症及死亡率一致，依此結果推測，體顯性多囊腎病患的心血管併發症和突變基因相關，而其致病機轉及未來治療仍待進一步研究。
人類的疾病非常複雜，身體內的器官系統互有交通也都相互影響，因此，同一疾病在不同器官的特定細胞到底如何致病，因果通常不易釐清，況且人體不同組織細胞的取得更是研究上的難題。未來將利用體顯性多囊腎病患幹細胞多潛能的特性，預計分化成不同細胞，研究體顯性多囊腎在不同器官系統的致病機轉，期望未來對多囊腎的疾病治療提供藥物篩選平台以發展創新有效的治療。

關鍵字

體顯性多囊腎 ； 人類誘導性多潛能幹細胞 ； 心臟血管併發症 ； 心律不整

並列摘要

Taiwan has the highest incidence and prevalence of end stage renal disease worldwide. Autosomal dominant polycystic kidney disease (ADPKD) is the 4th leading cause of renal failure, and is the most prevalent monogenic kidney disorder. Patients of ADPKD develop multiple kidney cysts and have progressive renal failure. Cardiovascular complication is the major cause of ADPKD patient mortality. Whether the kidney disease is directly or indirectly linked to the heart is still elusive. To date, no effective therapy has been developed for ADPKD.
Accumulating evidences shows the disease causative genes of ADPKD, PKD1 and PKD2, and their encoding proteins, polycystine 1 and polycystine 2, altered cardiomyocytes function in PKD animal models. In this research, my hypothesis was that the ADPKD mutation genes per se lead to the functional changes in cardiomyocytes and result in clinical ADPKD patients’ cardiovascular complications. Considering the differences between animals and human species regarding intracellular enzyme system and ion channels, I chose to apply patient-derived induced pluripotent stem cells (iPSCs) for use in this research.
Three iPSC lines were generated from selected ADPKD patients. I then differentiated the iPSCs toward ventricular-like cardiomyocytes. The calcium image study showed altered calcium handling of the ADPKD iPS cells-derived cardiomyocytes. Further electrophysiological experiments with whole cell-patch clamping revealed a remarkable similarity of spontaneous beating pattern and drug responsiveness of the ADPKD iPS cells-derived cardiomyocytes with their donors. More importantly, ADPKD iPS cells-derived cardiomyocytes had proarrhythmia characteristics elicited by beta-adrenergic agonists. Taken together, the results from the ADPKD patient-specific iPS cells, in line with the animal studies, suggested possible direct links between the mutation genes and the cardiovascular complications of ADPKD patients.
Human cell-based disease modeling in vitro is invaluable for studying species-specific mechanisms, deciphering multisystem-involved human diseases, and as a drug screening platform. The novel results achieved by applying the ADPKD patient-specific iPSCs pave the way to a more extensive and comprehensive study of cardiovascular complication in ADPKD patients, especially focusing on cardiac arrhythmia. In the future, I will continue investigating the molecular mechanisms of the mutation genes leading to the cellular phenotype in cardiomyocytes, and to develop effective novel therapies for ADPKD patients.

並列關鍵字

Autosomal Dominant Polycystic Kidney Disease ； Human induced pluripotent stem cell ； cardiovascular complication ； arrhythmia

參考文獻

References

1 United States Renal Data System. 2018 USRDS annual data report: Epidemiology of kidney disease in the United States. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, 2018.

2 台灣腎病年報. 2017 / 許志成總編輯. -- 第一版. -- 苗栗縣竹南鎮 : 國家衛生研 究院 ; [臺北市] : 台灣腎臟醫學會, 民 107.06

3 Ong ACM, Devuyst O, Knebelmann B, and et al. Autosomal dominant polycystic kidney disease: the changing face of clinical management. Lancet 2015; 385: 1993-2002.

4 Wilson PD, Goilav B. Cystic disease of the kidney. Annu Rev Pathol 2007; 2:341-68.