臺灣博碩士論文加值系統

腎臟纖維化乃進行性慢性腎臟病的最終結果。未緩解的過度腎臟發炎反應導致製造細胞外基質的細胞過度活化與增生，因而導致腎臟纖維化。愈來愈多的證據顯示在腎臟纖維化中，巨噬細胞從促發炎的M1亞型轉為具抗發炎與組織修補能力的M2亞型之現象至關重要。然而，在腎臟纖維化中調控M1/M2 亞型平衡的因子尚未完全被了解。由骨髓衍生的間葉幹細胞為多功能幹細胞，能夠藉由感測發炎環境中的訊息來調控免疫反應以及巨噬細胞極化現象的方式達到促進組織修復的功能。缺氧處理間葉幹細胞在單側輸尿管結紮小鼠模式中相較於常氧間葉幹細胞具有較佳之組織修復能力，但詳細機轉為何目前並不清楚。於本研究中，利用單側輸尿管結紮小鼠之腎臟裂解液刺激J774細胞株成為似M1巨噬細胞的體外模式來模擬腎臟纖維化，我們發現缺氧處理間葉幹細胞較常氧間葉幹細胞更能有效促進似M1巨噬細胞的mannose receptor與arginase 1之表現，並抑制誘導型一氧化氮合成酶（iNOS）的信使核糖核酸與蛋白質表達。有趣的是在發炎環境下，缺氧處理間葉幹細胞本身的iNOS表現量與一氧化氮（NO）的產量會增加，事先以L-NAME （iNOS抑制劑）處理缺氧處理間葉幹細胞之後，缺氧處理間葉幹細胞之促M2極化現象能力則顯著減弱; 加入SNAP（NO產生劑）後則可直接促使似M1巨噬細胞轉成M2表型。綜合以上，本研究發現在體外腎臟纖維化模式中，缺氧處理間葉幹細胞可上調iNOS表現，進而增加NO的釋放量來促進巨噬細胞之M2極化現象。我們的發現提供實驗室的佐證：缺氧處理間葉幹細胞未來將有潛力可治療臨床發炎性腎纖維疾病。

Renal fibrosis is the common final consequence of progressive chronic kidney diseases. Non-resolving inflammation triggers the excessive activation and expansion of matrix-producing cells, which ultimately lead to renal fibrosis. Mounting evidence suggests that the phenotypic switch of macrophages from a pro-inflammatory M1 phenotype to anti-inflammatory, reparative M2 ones critically governs renal fibrosis. Nonetheless, regulators of the M1/M2 balance in renal fibrosis remain incompletely understood. Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent stem cells that can enhance tissue regeneration by tempering immune responses and macrophage polarization in response to the inflammatory microenvironment. Hypoxia-preconditioned MSCs (hMSCs) have greater tissue repairing capability in unilateral ureteral obstructed (UUO) mice with renal fibrosis. Whether hMSCs improve renal fibrosis in UUO via modulation of macrophage phenotypes is still elusive. Herein, to mimic the inflammatory microenvironment in renal fibrosis, we first utilized UUO-kidney lysate (UUO-KL) to establish M1-polarized J774 macrophages. Furthermore, hMSCs increased mannose receptor and arginase 1 expressions and decreased inducible nitric oxide synthase (iNOS) expression in co-cultured J774 macrophages as compared to normoxia-cultured MSCs. Interestingly, the expression levels of iNOS and nitric oxide (NO) in hMSCs per se were upregulated by the pro-inflammatory milieu. Pre-treatment with L-NAME (an iNOS inhibitor) significantly suppressed the M2-promoting effect of hMSCs, while administration of SNAP (an NO donor) skewed M1-J774 macrophages toward M2 phenotypes. Taken together, the present study found that hMSCs promoted an M1-to-M2 macrophage polarization in UUO, possibly through upregulating iNOS expression and augmenting NO production. Our findings provided the bench evidence to support that hMSCs could be a potential therapeutic approach for inflammatory fibrotic kidney diseases in the future.

Contents
Acknowledgement.......i
中文摘要.......iii
Abstract.......iv
Contents.......v
List of tables.......vii
List of figures.......viii
1. Introduction.......1
1-1. Chronic Kidney Disease and Renal Fibrosis.......1
1-2. Unilateral Ureteral Obstruction (UUO) Model and Macrophage Polarization.......2
1-3. Mesenchymal Stem Cells (MSCs) and Macrophage Polarization.......3
1-4. Hypoxic Mesenchymal Stem Cells (hMSCs) and Renal Fibrosis.......3
2. Hypothesis and Specific Aims of this Study.......4
3. Materials.......5
3-1. Animals.......5
3-2. Cells.......5
3-3. Cell Culture Media.......5
3-4. UUO-KL Preparation.......6
3-5. Cell Coculture System.......6
3-6. RNA Purification and Reverse Transcription.......6
3-7. Real-Time PCR for mRNA Detection.......6
3-8. Western Blotting.......6
3-9. Flow Cytometry.......7
3-10. Enzyme-Linked Immunosorbent Assay (ELISA).......7
3-11. Griess Reaction for Nitrite Detection.......7
3-12. NO Donor.......8
3-13. iNOS Inhibitor.......8
3-14. Lentivirus Construction.......8
4. Methods.......9
4-1. Experimental UUO Animal Model.......9
4-2. UUO-KL Preparation.......9
4-3. J774 cells and MSCs/hMSCs Coculture Experiment.......9
4-4. Western Blotting Analysis.......9
4-5. RNA Isolation and Real-Time Quantitative Polymerase Chain Reaction.......10
4-6. Flow Cytometry.......10
4-7. ELISA Assay.......10
4-8. Griess Reation.......10
4-9. iNOS Inhibition.......11
4-10. Lentivirus Construction and Nos2 shRNA Transduction.......11
4-11. Statistical Analysis.......11
5. Results.......12
5-1. UUO-KL Polarized J774 Cells into a M1-like Macrophage.......12
5-2. hMSCs Exerted Earlier M2 Polarizing Effect on J774 Cells via Paracrine Effect.......12
5-3. Soluble-Factors-Based Crosstalk between hMSCs and UUO-KL-primed J774 Cells is Crucial to hMSCs-Modulated M2 Polarization.......12
5-4. NO is a Novel Molecule in hMSCs-Modulated M2 Polarization.......13
5-5. Extracellular Signal-Regulated Kinase (ERK) and Mitogen-Activated Protein Kinase Phosphatase-1 (MKP-1) Pathways were Activated in hMSCs-Derived-NO-Directed M2 Polarization.......14
6. Discussion.......15
7. References.......18

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