臺灣博碩士論文加值系統

非酒精性脂肪肝（NAFLD）為常見之代謝性疾病。目前文獻報導指出人類間充質幹細胞（MSCs）能透過分泌生長因子等具生物活性成分以抑制免疫反應或加速組織修復。本研究利用MSC-based及MSC lysate-based兩種策略對NAFLD進行治療。其實驗組別於MSC-based組包含MSC、MSC分化之棕色脂肪細胞、MSC每日注射三種；MSC lysate-based組包含MSC lysate、MSC+MSC lysate兩種。研究結果指出，本實驗所用的飲食誘導之NAFLD動物模型，其疾病進程在兩策略各實驗組中均有明顯的回復，包含了Oil red O及天狼星紅染色結果，及各項與肝臟再生、纖維化、發炎反應有關的基因表現等。同時本研究也發現前述治療效果與血液中脂聯素（adiponectin）濃度及下游訊息傳導有關。進一步本研究也發現MSC-based治療策略能顯著增加血液HDL/LDL比值；MSC lysate-based治療策略能顯著增加實驗動物之葡萄糖耐受性及降低血液中GOT及GPT濃度。總結以上，本研究結果指出無論MSC或MSC lysate皆對於治療NAFLD有極大治療潛力。

Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder. Recently, human mesenchymal stem cells (MSCs) have been reported to reduce inflammatory response and promote tissue regeneration through secretion of growth factors. Here we provide two therapeutic strategies for treating NAFLD by using MSC-based or MSC lysate-based approach. Results showed diet-induced NAFLD were ameliorated in our mice model in either MSC-based or MSC lysate-based approaches based on liver Oil red O and Sirius red stain results, and different extends of acceleration of regeneration, reversion of fibrosis and reduction of inflammation were also showed in the liver. And we also discovered adiponectin-induced correction of metabolic disorder in either approaches. Besides, MSC-based approach can also restored the ratio of HDL/LDL; MSC lysate-based approach can increased glucose tolerance and provided hepatoprotection effect by reducing serum GOT and GPT level. These results showed that MSC-based and MSC lysate-based therapeutic approaches are strong potential candidates for NAFLD therapy.

Contents
中文摘要 i
Abstract ii
Contents iii
List of Tables v
List of Figures v
Abbreviation vii
Introduction - 1 -
1.1. Non-alcoholic fatty liver disease - 1 -
1.2. Mesenchymal stem cells - 5 -
1.3. Brown adipose tissue - 7 -
1.4. Motivation - 10 -
Materials and Methods - 11 -
1.5. Cell culture - 11 -
1.5.1. Culture maintenance and cell expansion - 11 -
1.5.2. Cell lysate preparation - 11 -
1.5.3. Brown adipocyte differentiation of MSCs - 12 -
1.6. Animal experiments - 12 -
1.6.1. Non-alcoholic fatty liver disease animal model - 12 -
1.6.2. Serum sample collection and preparation - 13 -
1.6.3. Tissue sample preparation for cryosection - 13 -
1.6.4. RNA sample preparation - 14 -
1.6.5. Intraperitoneal glucose tolerance test (IPGT test) - 14 -
1.6.6. Serum biochemical analysis - 15 -
1.6.7. Experiment group classification - 15 -
1.7. In Vitro analysis of disease status - 16 -
1.7.1. H &; E staining - 16 -
1.7.2. Oil red O staining - 16 -
1.7.3. Sirius red staining - 17 -
1.7.4. RT-PCR and qPCR - 17 -
1.7.5. Enzyme-linked immunosorbent assay (ELISA) - 18 -
Results - 19 -
1.8. Long term high fat diet feeding induces type 3 fatty liver disease. - 19 -
1.9. Paracrine effect improves brown adipocyte differentiation. - 21 -
1.10. Treatment strategies and group classification. - 22 -
1.11. Diet-induced obesity, hepatomegaly and hepatosteatosis were reversed by different extends in both approaches. - 22 -
1.12. Cell transplantation can increases HDL/LDL ratio and lysate injection can provide hepatoprotection in NAFLD animals. - 24 -
1.13. Different extends of mitogen response induced liver regeneration and maturation were induced by each approaches. - 25 -
1.14. MSCs had higher ability of correcting high fat diet-induced inflammation and ECM abnormality. - 27 -
1.15. Adiponectin up-regulation might be the cause of therapeutic effect in our model. - 30 -
1.16. Peroxisome proliferator-activated receptor α and γ might be the key factors of reversing the status of HFD induced metabolic syndromes. - 30 -
1.17. The change of glucose transporter 4 shows the same trend with Pparγ in fat and muscle. - 33 -
1.18. Lysate injection dramatically improved the glucose tolerance. - 34 -
1.19. Different extends of reduction of T2D markers were exhibited in experiment groups. - 34 -
Discussion - 36 -
Conclusion - 39 -
Perspective - 40 -
References - 41 -
Tables - 48 -
Figures - 50 -
List of Tables
Table 1. Sequences of mouse qPCR primers - 48 -
List of Figures
Figure 1. Animal weight change during 30 weeks feeding. - 50 -
Figure 2. High fat diet induce obesity, liver steatosis and Glc intolerance in mice - 52 -
Figure 3. High fat diet causes expression change of several serum markers. - 54 -
Figure 4. Partial media change enhance beige adipocyte differentiation efficiency. - 57 -
Figure 5. Schematic illustration of the experiment approaches. - 58 -
Figure 6. The animal weight and the liver size change induced by either cell-based or lysate-based approaches. - 59 -
Figure 7. Oil red O stain results from each experiment groups. - 61 -
Figure 8. Serum biochemical analysis results from cell-based approach groups animals. - 63 -
Figure 9. Serum biochemical analysis results from lysate-based approach groups animals. - 65 -
Figure 10. Expression change of liver mitogen response, regeneration and maturation genes in cell-based approach groups. - 67 -
Figure 11. Expression change of liver mitogen response, regeneration and maturation genes in lysate-based approach groups. - 69 -
Figure 12. Expression change of pro-inflammatory, anti-inflammatory and fibrotic ECM genes in cell-based approach groups. - 71 -
Figure 13. Expression change of pro-inflammatory, anti-inflammatory and fibrotic ECM genes in lysate-based approach groups. - 73 -
Figure 14. Sirius red stain results from each experiment groups. - 75 -
Figure 15. Serum adiponectin expression change in both cell-based and lysate-based approaches. - 76 -
Figure 16. Expression change of liver Pparα and Pparγ gene expression levels in both cell-based and lysate-based approach groups. - 77 -
Figure 17. Expression change of fat Pparα and Pparγ gene expression levels in both cell-based and lysate-based approach groups. - 78 -
Figure 18. Expression change of muscle Pparα and Pparγ gene expression levels in both cell-based and lysate-based approach groups. - 79 -
Figure 19. Expression change of pancreas Pparα and Pparγ gene expression levels in both cell-based and lysate-based approach groups. - 80 -
Figure 20. Expression change of Glut4 gene expression in fat and muscle in both cell-based and lysate-based approach groups. - 81 -
Figure 21. IPGT test results in both cell-based and lysate-based approach groups. - 82 -
Figure 22. Expression change of T2D marker genes in liver in both cell-based and lysate-based approach groups. - 83 -

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