臺灣博碩士論文加值系統

背景: 腎骨病變嚴重威脅慢性腎臟病 (CKD) 患者的健康。在腎骨病變組織學的分類當中，低骨更替率疾病正變得越來越普遍。臨床研究證實長期血液透析患者血清中的尿毒素:硫酸吲哚酚 (IS) 和骨特異性鹼性磷酸酶呈現負相關，顯示IS與骨形成減少有關。動物實驗也顯示IS抑制骨形成。然而，很少有體外研究使用非細胞毒性濃度的IS觀察其是否影響骨髓間質幹細胞 (BMSCs) 的骨分化。同時，IS影響BMSCs骨生成的分子機制需要進一步研究。
方法: 本研究的D1細胞屬於小鼠BMSCs。使用MTT細胞活性測試和乳酸脫氫酶試驗來探討IS的非細胞毒性濃度。評估IS對D1細胞骨分化的影響則使用即時定量聚合酶連鎖反應觀察骨生成相關基因的表現，以及使用茜素紅S鈣染色法評估D1細胞骨分化時的礦化情形。
結果: MTT細胞活性測試的研究結果顯示濃度介於100─400、75─400、以及75─400 μM (P < 0.05、P < 0.01、以及P < 0.05) 的IS抑制D1細胞的存活。乳酸脫氫酶試驗顯示濃度介於75─400 μM 的IS在細胞培養的第一、二、三天對於D1細胞具有毒性 (P < 0.05)。將濃度介於 25─400 μM 的IS用以測試D1細胞的骨分化。DNA定量測試的結果顯示D1細胞在骨分化時的細胞數量不受IS所影響。濃度為25以及50 μM的IS，於細胞增殖時不影響D1細胞存活，但是在D1細胞接受成骨誘導後，能抑制其在第七與第十天的骨分化 (P < 0.01)，同時不影響細胞的數量。在骨生成相關基因的表現上，濃度介於 50─200 μM 的IS降低骨分化早期的骨塑型蛋白二的表現 (P < 0.05)，而濃度介於 25─200 μM 的IS則降低骨分化晚期鹼性磷酸酶以及骨鈣素基因的表現 (P < 0.01)。
結論: IS (25─50 μM)不影響細胞存活，但能降低BMSCs的骨分化。此濃度區間的IS符合慢性腎臟病人的平均血中濃度。因此，本研究結果推測IS是引起慢性腎衰竭病人低骨更替率疾病的重要因子。

Background: Renal osteodystrophy is a serious health concern for patients with chronic kidney disease (CKD). Low turnover bone disease, which is one of the histological categories of renal osteodystrophy, are becoming more prevalent. Clinical studies have proven the negative correlation between the serum levels of uremic toxin indoxyl sulfate (IS) and bone-specific alkaline phosphatase in chronic hemodialysis patients, which indicates that IS relating to the decrease of bone formation. In vivo studies have also demonstrated that IS inhibits bone formation. However, few in vitro studies evaluated osteogenic differentiation using a non-cytotoxic concentrations of IS in bone marrow-derived mesenchymal stem cells (BMSCs). Accordingly, the molecular mechanisms of IS affecting osteogenesis in BMSCs requires further investigation.
Methods: D1 cells are mouse BMSCs and were used for this study. MTT assay and lactate dehydrogenase assay were used to search the non-cytotoxic concentrations of IS. The effect of IS on osteogenic differentiation of D1 cells was evaluated by osteogenic gene expression using quantitative real-time polymerase chain reaction and mineralization using alizarin red S staining.
Results: The results of IS affecting D1 cells viability showed that
IS at concentrations of 100 to 400, 75 to 400, and 75 to 400 μM significantly decreased MTT activity (P < 0.05, P < 0.01, and P < 0.05, respectively) and at concentrations of 75 to 400 μM elevated lactate dehydrogenase leakage of D1 cells at 1, 2, and 3 days (P < 0.05) after starting the cell culture, respectively. The IS at concentrations of 25 to 400 μM was used to test osteogenic differentiation of D1 cells. The results showed that the quantity of D1 cells which was measured by DNA quantification assay was not affected by IS at concentrations ranging from 25 to 400 μM during osteogenic differentiation. IS at concentrations of 25 and 50 μM, which did not affect the viability of D1 cells during proliferation, reduced osteogenic differentiation without influencing cell quantity at 7 and 10 days (P < 0.01) after osteogenic induction. In mechanistic studies, IS at concentrations of 50 to 200 μM (P < 0.05) downregulated BMP-2 expression during the early stages of osteogenic differentiation, and IS at concentrations of 25 to 200 μM (P < 0.01) downregulated ALP and OC expression during the late stages of osteogenic differentiation.
Conclusions: In this study, we found that IS of 25 to50 μM reduced osteogenic differentiation of BMSCs without influencing cell viability. The effective concentrations of IS found in this study are at the average serum concentrations of IS in patients with CKD. From this finding, we suggest that IS is a crucial factor contributing to low bone turnover in patients with CKD.
Abbreviation:
MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide
BMP-2: bone morphogenetic protein 2
ALP: alkaline phosphatase
OC: osteocalcin

TABLE OF CONTENTS
INTRODUCTION AND OVERVIEW 1
Effects of uremic toxins on the bone 2
1. Indoxyl sulfate (IS) 2
2. Uremic toxins may reduce the rate of bone formation 3
3. Unclear molecular mechanism of uremic toxins on bone formation 3
RESEARCH METHODOLOGY 5
Cell culture 5
Cell viability and cytotoxicity test 6
1. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay 6
2. Lactate dehydrogenase (LDH) leakage 6
Osteogenic differentiation of bone marrow mesenchymal stem cells 6
1. Osteogenic gene expression by real-time polymerase chain reaction 7
2. DNA quantification 8
3. Alizarin red S staining for mineralization 8
Statistical analysis 9
RESULTS 10
Effects of IS on cell viability 10
mRNA expression analysis of bone formation-related genes using real-time PCR 10
Effects of IS on osteogenic differentiation of BMSCs 11
DISCUSSION 28
CLINICAL RELEVANCE 31
REFERENCES 32

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