臺灣博碩士論文加值系統

末期腎病變(End-Stage Renal Disease，ESRD)的盛行率及發生率在台灣及世界持續攀升，已經成為重要的社會醫療議題。當慢性腎臟病 (Chronic Kidney Disease, CKD) 進展成為末期腎病變的過程中，患者因腎功能衰退，而堆積身體產生代謝廢物。文獻指出：部份代謝廢物會影響正常器官功能，因此被稱為尿毒素 (Uremic toxins)。其中，硫酸吲哚酚 (Indoxyl Sulfate, IS) 是蛋白質中的胺基酸tryptophan 經腸道細菌及肝臟代謝而產生，為一種親蛋白的尿毒素，主要經由腎臟排出體外。CKD患者因腎功能衰退，無法排除體內的IS，而累積於體內。而肌少症 (Sarcopenia) 是指肌肉的質量、肌力、耐力減少的現象，是一種老化及退化症狀。 Sarcopenia 對末期腎病變 患者影響包括加速器官功能退化、容易跌倒、甚至死亡。MyoD、Myogenin和Myosin heavy chain (MHC)，為骨骼肌細胞分化重要的biomarker，其中MyoD和Myogenin皆屬於肌肉發育調控因子（Myogenic Regulatory Factor，MRF)，為transcription factors，在肌肉分化過程中會依序出現。MHC是細胞骨架的一種，在肌肉收縮過程中扮演著重要的角色。過去有文獻指出：尿毒素會影響心肌細胞功能，對骨骼肌的分化影響則未著墨。本研究使用小鼠骨骼肌細胞母細胞株 (C2C12 myoblast)探討 IS 對骨骼肌細胞分化的影響。本研究主要分為三個部分：(1) IS在生長培養液處理C2C12 myoblast 72小時，探討IS對其母細胞株的增生及毒性分析；(2) C2C12 myoblast在生長培養液中暴露IS兩天，再於分化培養液中暴露IS四天，以探討IS對其母細胞株分化的影響；(3) C2C12分化四天後再處理IS 72小時，以探討IS對其分化後的肌小管(myotube)的降解(degradation)的影響。本研究主要使用cell proliferation assay、flow cytometry、H&E stain、Western Blot 和 qPCR進行分析。研究結果顯示，IS 不會抑制C2C12增生，但會誘發ROS的產生以抑制AKT磷酸化，使分化明顯受到抑制，在Western Blot 結果顯示1mM的IS 會抑制MHC 和 Myogenin蛋白及RNA的表現，但MyoD的蛋白及RNA表現量未受到影響。因此，本研究後續將探討IS是否經由影響MyoD的功能進而減少下游蛋白Myogenin以及MHC的表現。本研究證實：尿毒素IS可以抑制骨骼肌肉母細胞分化，進而解釋末期腎病變患者出現高比例的肌少症的可能分子機轉之一。

End-Stage Renal Disease (ESRD) is highly incident and prevalent in Taiwan and the world. During the progression of chronic kidney disease (CKD) to ESRD, the metabolic wastes (uremic toxins) accumulate in the body leading to systemic organ dysfunction, including high cardiovascular events, immune dysfunction and cachexia. Indoxyl sulfate (IS), a protein-bound uremic toxin, is metabolized from indole in liver, which is metabolized from tryptophan by bacteria tryptophanase in the intestine. Sarcopenia is a phenomenon of reducing muscle mass, strength, and function, which can increase organ degeneration and mortality rates. Myosin heavy chain (MHC), Myogenin (MyoG) and myoblast determination protein 1 (MyoD) are important muscle skeleton differentiation biomarkers. MyoG and MyoD are transcription factors of myogenic regulatory factor (MRF). MHC is cytoskeleton, the major role of muscle contraction. Previously, there was the articles suggested that uremic toxins were able to disturb function of cardiomyocytes. However, it remains unclear whether the IS is associated with the skeleton muscle dysfunction. In this study, we investigated the potential effects of IS on the differentiation of C2C12 myoblast. We divided our study into three parts. (1) To explore the potential proliferation inhibition of C2C12 myoblast by IS (2) To study the inhibition of myoblast differentiation by IS (3) To investigate the degradation of C2C12 differentiated myotube by IS. We applied cell proliferation assay, flow cytometry, H&E stain, Western Blot and qPCR to analyze possible mechanisms. In Cell proliferation assay, we found that IS did not inhibit C2C12 myoblast cell proliferation. H&E stain, Western Blot and qPCR displayed that IS suppressed C2C12 myoblast differentiation. In order to explore the possible mechanisms, we demonstrated that IS did not suppress C2C12 myoblast proliferation, but IS induced reactive oxygen species (ROS) with following inhibition of AKT phosphorylation during C2C12 myoblast differentiation. As shown in the Western Blot, IS (1mM) inhibits the expression of MHC and Myogenin, but not MyoD, in the transcriptional and translational level. Furthermore, the degradation of differentiated C2C12 myotube did not induced by IS treatment. In conclusion, this study proved that IS suppressed skeletal myoblast differentiation, and successfully shown the possible molecular mechanisms, which might explain, at least partially, the high prevalent rates of uremic sarcopenia.

Chinese abstract.............................................i
Abstract...................................................iii
List of Abbreviation.........................................v
Chapter 1 Introduction.......................................1
1.1 Chronic Kidney Disease...................................1
1.2 Uremic toxins............................................2
1.3 Indoxyl sulfate..........................................2
1.4 The effect of Indoxyl sulfate............................3
1.5 Muscle wasting and Sarcopenia............................5
1.6 Skeletal muscle differentiation..........................7
1.7 Myogenic regulatory factors (MRFs).......................8
1.8 Aim of this study.......................................10
Chapter 2 Materials and Methods.............................11
2.1 Flowchart...............................................11
2.2 Cell culture............................................12
2.3 Cell proliferation assay................................13
2.4 Cell viability assay....................................14
2.5 Hematoxylin and eosin (H&E) staining....................15
2.6 Myotube nuclear calculation.............................15
2.8 Protein extraction and immunoblotting...................16
2.9 RT-qPCR analysis........................................18
2.10 Statistical analysis...................................20
Chapter 3. Result...........................................21
3.1 IS exposure inhibited mytube formation, but did not affect cell viability..............................................21
3.2 C2C12 myoblast expose IS 0 to 1mM does not influence proliferationy..............................................21
3.3 IS decreased myogenic specific genes expression during muscle differentiation......................................22
3.4 IS enhanced TGF-β1 expression...........................23
3.5 IS induce ROS on C2C12 myoblast.........................23
3.6 Using a ROS inhibitor, NAC, replied skeletal muscle differentiation with 1mM IS treatment.......................24
3.7 IS inhibited myoblast differentiation through ROS.......25
3.8 IS could not induce atrophy on C2C12 myoblast...........25
Chapter 4. Discussion.......................................26
Chapter 6. Future work......................................31
Chapter 7. References.......................................32
Chapter 8. List of Tables and Figures.......................43
Figure I. Metabolism of Indoxyl sulfate and effect of AST-120 .............................................................3
Figure II. Possible aetiology of uraemic sarcopenia..........6
Figure III. The modal of MyoD in skeletal muscle.............9
Table1. Antibody for western blot...........................18
Table 2. C2C12 Primers for real-time PCR....................19
Figure 1....................................................43
Figure 2....................................................46
Figure 3....................................................48
Figure 4....................................................51
Figure 5....................................................53
Figure 6....................................................55
Figure 7....................................................57
Figure 8....................................................59
Supplementary figure 1......................................61

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