臺灣博碩士論文加值系統

因動脈閉塞所引起的嚴重肢體缺血（ CLI ）常導致肢體壞死而必須截肢。治療肢體缺血的有效方法之一是將幹細胞直接注射到缺血的組織進行治療。多能間質細胞，也稱為間葉幹細胞（MSCs），為一種具多能分化，便於分離純化，可於體外放大培養的細胞來源，並能有效應用於自體的細胞治療。骨髓間葉幹細胞已經被應用在血管新生，能夠幫助新血管和肌肉細胞形成與修復。然而，骨髓間葉幹細胞作為肢體缺血疾病的異體移植的治療效果仍然存在著爭議。找出使骨髓間葉幹細胞減少產生免疫反應的方法，可以幫助骨髓間葉幹細胞更有效的應用於治療心血管疾病。骨髓間葉幹細胞培養在低氧條件下，可減少細胞增殖性衰老，增加增殖率，提高分化潛能以及移植後的存活能力。在本研究中，我們證明相較於含氧量正常下培養的骨髓間葉幹細胞，在低氧培養之C57BL/6J (B6)小鼠骨髓間葉幹細胞可顯著改善BALB / c小鼠的肢體缺血情形。然後我們證明低氧骨髓間葉幹細胞增加異體細胞移植的存活能力，降低自然殺手（NK）細胞毒殺活性，並降低在體內移植時來自宿主自然殺手細胞的積聚。這些異體骨髓間葉幹細胞增加了缺血肢體中CD31內皮細胞與SMA和desmin肌肉細胞的產生，進而促進了血管生成和恢復肌肉的結構。此外，當以抗體中和自然殺手細胞時，能增加常氧骨髓間葉幹細胞於異體缺血肢體的血管生成能力並避免肢體截肢。這些結果強烈表明低氧骨髓間葉幹細胞可能作為治療心血管疾病的“通用供體細胞”。

Critical limb ischemia (CLI) caused by arterial occlusion often leads to limb necrosis and amputations. One effective therapeutic approach has involved the local injection of stem cells directly into ischemic tissue. Multipotent stromal cells, also termed mesenchymal stem cells (MSCs) are multipotential, ease to isolate, and amenable to ex vivo expansion which represent a promising cell source for ischemic limb autologous cell therapy. MSCs have been applied in neoangiogenesis via incorporation into newly formed vessels and muscle cells. However, the therapeutic effects of allogeneic transplantation with MSCs in limb ischemic diseases remain controversial. Identification of methods for enhancing immunoprivilege properties can facilitate the application of MSCs in treating cardiovascular diseases. MSCs cultured under hypoxic conditions decreased in replicative senescence, increased in proliferation rate, differentiation potential and the ability of engraftment after transplantation. In this study, we show that hypoxic MSCs from C57BL/6J (B6) mice increase in ameliorating limb ischemia of Balb/c mice compared to normoxic MSCs. We then demonstrate that hypoxic MSCs increase the ability to engraft in allogeneic recipients, reduce natural killer (NK) cells cytotoxicity, and decrease accumulation of host-derived NK cells when transplanted in vivo. These allogeneic hypoxic MSCs give rise to CD31+ endothelial cells and SMA+ and desmin+ muscle cells, thereby enhance angiogenesis and restore muscle structure. Moreover, normoxic MSCs when neutralized with anti-NK cell antibodies increased in enhancing angiogenesis and preventing limb amputation in allogeneic recipients with limb ischemia. These results strongly suggest that hypoxic MSCs may serve as a “universal donor cell” for treating cardiovascular diseases.

Introduction 1
Study purpose 3
Specific aims 3
Methods 5
MSCs isolation, characterization and culture 5
Flow cytometry analysis 6
Differentiation assays 6
Ischemic limb models 7
Evaluation of limb perfusion 8
Immunohistochemistry and immunofluorescence 8
NK cell cytotoxicity assay 9
Statistical analysis 10
Results 11
Characterization of MSCs cultured under hypoxic and normoxic conditions 11
Allogeneic transplantation of hypoxic MSCs increases limb ischemia improvement compared to normoxic MSCs 11
Hypoxic MSCs decrease muscle degeneration and fibrosis and increase the angiogenesis of the ischemic limb 13
Hypoxic MSCs survive and engraft after transplantation 15
Hypoxic MSCs decrease in NK cells accumulation in allogeneic recipients 16
Hypoxic MSCs escape NK cells lysis 17
Discussion 19
References 24


List of Figures
Figures 29
Figure 1. Surface marker characteristics of normoxic and hypoxic MSCs. 30
Figure 2. Differential potency of normoxic and hypoxic MSCs. 33
Figure 3. Hypoxic MSCs increase in the expression of several HIF-1α regulated factors. 34
Figure 4. Hypoxic MSCs increase in the expression of several immune tolerance related factors. 35
Figure 5. Hypoxic MSCs improve limb ischemia in allogeneic recipients. 36
Figure 6. Hypoxic MSCs improve limb perfusion in allogeneic recipients. 38
Figure 7. Hypoxic MSCs decrease the muscle degeneration as well as fibrosis and increase the blood perfusion of ischemic limb in allogeneic recipients. 43
Figure 8. Short-term survival of hypoxic MSCs after allogeneic transplantation into ischemic limbs. 45
Figure 9. Hypoxic MSCs decrease in apoptosis in vivo. 46
Figure 10. Long-term engraftment of hypoxic MSCs after allogeneic transplantation into ischemic limbs. 47
Figure 11. Long-term engraftment of hypoxic MSCs after allogeneic transplantation into ischemic limbs induce vascular and muscular networks in the ischemic muscles. 50
Figure 12. Long-term engraftment of hypoxic MSCs by detection of genetic locus. 53
Figure 13. Hypoxic MSCs decrease in NK cells accumulation in allogeneic recipients. 56
Figure 14. Hypoxic MSCs do not accumulate CD8+ or CD4+ T cells, CD14+ macrophage, CD86+ dendritic cells, or TCRαβ+ NKT cells in allogeneic recipients. 58
Figure 15. Neutralization of normoxic B6 MSCs with anti-NK ligand antibodies enhances their effect in enhancing blood perfusion while restoring muscle structure in ischemic limbs. 60
Figure 16. Hypoxic MSCs escape NK cells lysis in vitro. 61
Figure 17. Allogeneic NK cells-mediated lysis was observed in human normoxic MSCs but not in human hypoxic MSCs. 62

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