臺灣博碩士論文加值系統

顯微⽪瓣重建⼿術是整形外科⼿術中治療重要器官、組織外露及⽪膚缺損的必要⼿術，但⽪瓣壞死卻是顯微⼿術經常遇到的後遺症，⼀旦⽪瓣壞死將會造成患者及醫師極⼤的困擾，甚⾄危及⽣命；因此，如何增加⽪瓣⼿術的成功率將是⼀個重要的議題。本研究⽬標為：釐清脂肪衍⽣幹細胞及其衍⽣物是否能藉由⾎管新⽣作⽤保護經缺⾎再灌流⽽受損的⽪瓣，並增加其存活率。實驗設計為：將供應⼩⿏腹側⽪瓣的⾧胸⾎管(long thoracic vessels)以⾎管夾緊夾住三⼩時後再放開，以使⾎液流⼊⽪瓣，同時直接注射脂肪衍⽣幹細胞、幹細胞液態培養基(conditioned medium)及其外吐⼩體(exosome)到⽪瓣內，並與未治療之組別⽐較⽪瓣之活率。實驗結果發現，以脂肪衍⽣幹細胞、幹細胞液態培養基及其外吐⼩體治療之組別，在術後五天之⽪瓣存活率⾼於未治療之組別，且有顯著之差異。無論細胞裂解液(cell lysates)或細胞液態培養基中，脂肪衍⽣幹細胞介⽩素-6 的量均⽐⼈類纖維母細胞(Hs68)來得多；並且脂肪衍⽣幹細胞之液態培養基及其外吐⼩體增加了⾎管管柱的形成。我們如果⽤中和介⽩素-6 的抗體或⼩分⼦干擾核糖核酸(Si-IL6-ADSC)治療受損的⽪瓣，則無法得到治療效果。使⽤脂肪衍⽣幹細胞治療⽪瓣受損的介⽩素-6 基因剔除⼩⿏(IL-6 knockout mice)亦可增加其⽪瓣存活率。此外，我們發現，介⽩素-6 的產⽣和新⽣⾎管作⽤是由傳統訊息傳遞途徑(classic-signaling pathway)並活化訊息傳遞轉錄活化基因(signaltransducer and activator of transcription)作⽤。由實驗得知，增加⽪瓣存活率的機轉包括：脂肪衍⽣幹細胞直接分化成⾎管內⽪細胞增加新⽣⾎管及由脂肪衍⽣幹細胞釋放出之介⽩素-6 的間接作⽤；並且幹細胞液態培養基及其外吐⼩體可能可以以“現貨供應”的⽅式提供⽪瓣的治療。

Skin flap transplantation is the most widely used in plastic and reconstructive surgery. Unfortunately, flap necrosis is the most frequent postoperative complication encountered in reconstructive surgery. Thus, determining how to increase pro-angiogenic factor levels and augment angiogenesis plays an essential role in improving the survival of skin flaps and increasing the success rate of skin flap transplantation. Cell-based therapy with stem cells was a promising option for ischemia/reperfusion (I/R) injury. Mesenchymal stem cell (MSC) is one of the postnatal adult stem cells and possessed capacity for self-renewal and differentiation with a broad tissue distribution. Adipose-derived stem cells (ADSCs) is one of MSC with less ethical concern. We elucidated whether adipose-derived stem cells (ADSCs) and their derivatives might induce neovascularization and protect skin flaps during ischemia/reperfusion (I/R) injury. The skin flap was subjected to 3 h of ischemia by ligating long thoracic vessels and then to blood reperfusion. Q-tracker-labeled ADSCs, conditioned media (ADSC-CM), or exosomes (ADSC-Exo) from ADSCs were injected into the flap. These treatments led to a significant increase in flap survival and capillary density compared to I/R on postoperative day 5. Interleukin-6 (IL-6) is a pleiotropic cytokine that has a broad spectrum of biological activities such as regulation of inflammation, cell proliferation, immunomodulation, haematopoiesis and tumorigenesis. It was reported to be a potent pro-angiogenic mediator in microenvironment. IL-6 levels either in the cell lysates or in CM were significantly higher in ADSCs than in Hs68 fibroblasts. ADSC-CM and ADSC-Exo increased EC tube formation. This result was corroborated by a strong decrease in skin repair after adding IL-6 neutralizing antibodies or si-IL-6-ADSC. ADSCs transplantation also increased flap recovery in I/R injury of IL-6 knockout mice. The IL-6 production and angiogenesis were accomplished through classic signaling pathway and the signal transducer and activator of transcription (STAT) activation. The mechanism of skin recovery includes both the direct differentiation of ADSCs into ECs and the indirect paracrine effect of IL-6 released from ADSCs. In the current study, we demonstrated that IL-6 in ADSCs, ADSC-CM and ADSCExo increased angiogenesis and enhanced recovery from I/R injury in the long thoracic artery pattern of skin flap in mice. The ADSC-CM and ADSC-Exo could be used as “off-the-shelf” products for this therapy.

口試委員會審定書
誌謝
中文摘要------------------------------------------------------------------------- I
英文摘要------------------------------------------------------------------------ III
Chapter 1. Introduction --------------------------------------------------------- 1
Chapter 2. Materials and Methods ---------------------------------------------- 6
2-1 Isolation and expansion of human adipose derived stem cells (ADSCs) ------ 6
2-2 Characterization of cultured ADSCs ---------------------------------------- 6
2-3 Characterization of the cells by immunocytochemistry ---------------------- 7
2-4 Differentiation of ADSCs into adipocytes, osteoblasts and chondrocytes ---- 7
2-5 Skin flap model and experimental design ----------------------------------- 8
2-6 Microvessel density --------------------------------------------------------10
2-7 Antibody array analysis ---------------------------------------------------- 10
2-8 Extraction, purification, and characterization of ADSC-exosomes ---------- 10
2-9 Western blot analysis ------------------------------------------------------ 11
2-10 IL-6 enzyme-linked immunosorbent assay (ELISA) in cell lysates
and conditioned media --------------------------------------------------- 12
2-11 siRNA transduction ------------------------------------------------------- 12
2-12 In vitro angiogenesis assay ----------------------------------------------- 13
2-13 Double immunofluorescence staining ------------------------------------- 13
2-14 Statistical analysis ------------------------------------------------------ 13
Chapter 3. Result
3-1 Characterization and pluripotent differentiation potential of ADSCs --------- 14
3-2 ADSCs transplantation enhances skin flap survival ------------------------- 14
3-3 ADSCs transplantation enhances the neovascularization of I/R injury
skin flap ------------------------------------------------------------------ 15
3-4 Abundant IL-6 in cell lysates and conditioned media from ADSCs
upregulate angiogenesis -------------------------------------------------- 16
3-5 IL-6 in ADSC-CM significantly promoted flap recovery by angiogenesis ---- 16
3-6 ADSC-Exo promoted flap recovery ----------------------------------------- 17
3-7 The phosphorylation of STAT3, but not ERK, was involved in
ADSCs-secreted IL-6 and angiogenesis ---------------------------------------- 18
Chapter 4. Discussion --------------------------------------------------------- 20
References ------------------------------------------------------------------- 24
Figure Contents and Legends
Figure 1. Morphology of ADSCs ------------------------------------------------ 31
Figure 2. Micrographs of hADSCs --------------------------------------------- 32
Figure 3. Flow cytometric analysis of ADSCs ----------------------------------- 33
Figure 4. ~ 6. Differentiation potential of ADSCs ------------------------------- 34
Figure 7. ADSCs transplantation enhances skin flap survival
after I/R operation --------------------------------------------------- 36
Figure 8. The skin flap recovery was evaluated by the histologic
scoring system ------------------------------------------------------ 38
Figure 9. ADSCs transplantation inhibits the decrease in
microvascular density of skin flap with I/R injury --------------------- 40
Figure 10. Representative images of Human Angiogenesis Antibody Array
and the corresponding lists of the tested angiogenic factors ---------- 43
Figure 11. Abundant IL-6 in cell lysates (CL) and conditioned medium (CM)
from ADSCs upregulates angiogenesis ------------------------------- 45
Figure 12. IL-6 induced tube formation of HUVECs ----------------------------- 47
Figure 13. IL-6 in ADSC-CM promoted flap recovery through angiogenesis ----- 48
Figure 14. The expression of IL-6 was much stronger in CM-, IgG- and
si-Scramble-treated I/R flap when compared with that in I/R,
anti-IL6- and si-IL6-treated I/R flap by immunohistochemistry -------- 52
Figure 15. ADSCs-Exo contained higher IL-6 levels and increased the flap
survival in I/R mice -------------------------------------------------- 53
Figure 16. The phosphorylation of STAT3, but not ERK,
was involved in ADSCs-secreted IL-6 and angiogenesis ------------- 56
Table Contents
Table 1. Summary of results from the angiogenesis antibody array ------------- 58

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