The Effect of Uremic Toxin Indoxyl Sulfate on Osteogenesis in Bone Marrow Mesenchymal Stem Cells
| Autor: | Chin-Wen Hsieh, 謝晉文 |
|---|---|
| Rok vydání: | 2018 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 106 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 |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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