臺灣博碩士論文加值系統

在過去,發現小鼠肺臟存在一群稀有的上皮幹源細胞，其表現胚胎幹細胞的細胞標記Oct-4、SSEA-1 以及 Sca-1與 Clara cell 的細胞標記 CCSP，以下簡稱mPSC (mouse pulmonary stem/progenitor cell)。本篇利用初級肺部組織細胞培養方法將 mPSC篩選出來。在細胞培養過程中，mPSC會形成colony 的形狀且其生長與幹性(stemness)的維持需要仰賴周圍的間質細胞(stromal cells)，一旦將mPSC 自細胞培養中分離出來至新的有第一型collagen覆蓋的培養皿中，將分化成第二型的肺泡細胞(type -2 pneumocytes)，之後繼續分化成第一型的肺泡細胞(type -1 pneumocytes)。成體幹細胞分佈於各種組織器官，在外來的損傷下能負責維持及修護特定組織之完整性。然而，我們對mPSC在外來的損傷刺激下所扮演的角色和機制仍不明，因此我們利用LPS刺激mPSC的模式，探討mPSC在損傷後所扮演的角色及其引發的機制。LPS全名為lipopolysaccharide，為革蘭氏陰性菌細胞壁外膜的成分之一,其常被拿來當作誘發肺部損傷或發炎的刺激物。由結果顯示，在LPS刺激mPSC之下, mPSC相較於周圍的stromal cells更具敏感性。首先，我們利用螢光免疫染色的方法發現mPSC 中的緊密接合蛋白,克奇沙病毒-腺病毒受體(CAR)，其表現有顯著的下降。另外,在LPS刺激後也發現趨化因子，MCP1 和 MIP1-α有明顯攀升的趨勢。而這兩個趨化因子屬於CC family的成員，主要功能為趨化單核球或巨噬細胞。另一方面，我們也發現到LPS對mPSC的刺激可能是透過其主要受體TLR4所引發。而在聚合酶連鎖反應分析下，發現到TLR4的表現有上升。於是，更進一步探究其下游所引發的訊息傳遞路徑，從西方點墨法顯示其活化路徑可能為ERK-MAPK 或NF-κB。另外，在mPSC與巨噬細胞的互動過程中，我們也更進一步地去分析巨噬細胞的分化情形。綜合以上的結果，我們認為在LPS刺激mPSC傷害模式下，mPSC可能具重要的免疫調節角色，且透過旁分泌效應來影響巨噬細胞。

In previous study, a rare population of lung epithelial stem/progenitor cells in mouse which expressed specific markers (Oct-4/SSEA-1/Sca-1/Clara cells secretion protein, CCSP), were identified and named mPSC. The cells could be enriched by a primary cells selection culture method. In the culture, mPSC could maintain in undifferentiated stage for weeks; and formed individual colonies with surrounding stroma cells. When the colonies of mPSC were plucked away from the cultures without stroma cells, and transferred to a new type-I collagen-coated culture dishes, the transferred cells were differentiated into type-2 and then type-1 pneumocytes in a sequential fashion. It is well known that tissue specific stem/progenitor cells play a role in repair and regeneration after tissue injury. However, the injury responses for the mPSCs were unclear. In this study, the primary culture was stimulated with LPS in order to reveal the injury mechanism and response for mPSCs. LPS is an outer membrane component of Gram-negative bacteria and often used as an inducer of lung injury. The results showed that mPSCs were more sensitive to LPS treatment comparing to stromal cell in the culture. In immunofluoresence staining analysis, LPS treatment down-regulated the expression of Coxsackievirus-adenovirus receptor (CAR), which is a tight junction protein of mPSCs. Additionally, the expression of chemokines, such as MCP1 and MIP1-α, was highly expressed after treatment. These CC chemokines are suggested to be associated with the interaction of macrophages. The results in this study also indicated that the stimulus of LPS for mPSCs was via toll-like receptor 4 (TLR4), the main receptor of LPS. The expression of TLR4 and its associated complex in mPSCs were analyzed by RT-PCR and Immunofluorescence staining respectively. Furthermore, the downstream signaling of TLR4, including NF-κB and MAPK pathways, were also examined by western blot. The results showed that TLR4 expression was up-regulated upon LPS stimulation and the LPS- induced injury pathway might be via the ERK-MAPK or NF-κB signaling pathway. In addition, we further show the polarization of macrophage in response to stimulation with various conditioned medium from mPSCs. Taken all together, we suggest that mPSCs may play a crucial role in regulating innate immune system upon LPS stimulation and exert paracrine effects to interact with macrophages.
 

Contents
Abbreviation I
中文摘要 II
Abstract III
Chapter 1. Introduction 1
1.1.Stem/progenitor cells 2
1.1.1. Stem/progenitor cells in the lung 2
1.1.2. Oct4+ pulmonary stem/progenitor cell in our culture system 3
1.2.Innate immune response in the lung 4
1.2.1. The role of macrophage in immune respons 4
1.2.2. Recognition of pathogens by airway epithelial cells 5
1.2.3. Toll-like receptors mediate pattern recognition signaling 6
1.2.4. Cytokines and Chemokines in inflammation 7
1.3.LPS-induced lung injury 9
1.4.LPS/TLR4 signaling pathway 10
1.5.Motivation 11
1.6.Aim 12
Chapter 2. Meterials and Methods 13
2.1.Cell culture 14
2.2.Cells stimulation with LPS 16
2.3.RNA extraction 16
2.4.Reverse transcription-polymerase chain reaction (RT-PCR)17
2.5.Immunofluorescence staining 17
2.6.Western blot 18
2.7.Migration assay 19
2.8.Cell viability assay 19
2.9.Propidium iodide (PI) staining 20
2.10.Statistics 20
Chapter 3. Results 21
3.1.The morphology of primary mPSC was changed after LPS stimulation 22
3.2.Cytotoxic effects of lung primary culture after LPS stimulation 22
3.3.The tight junction protein of primary mPSC was dissembling after LPS stimulation 23
3.4.Chemokine expression of primary mPSC after LPS stimulation 24
3.5.TLR4 receptor complex expression of primary mPSCs 26
3.6.LPS-induced activation of signaling pathways of primary mPSCs 27
3.7.The interaction between RAW 264.7 and mPSC in response to LPS 29
3.8.Modulation of macrophage polarization in response to different conditioned medium of mPSC 30
Chapter 4. Discussion 32
4.1.The signaling pathway of mPSC activated by LPS 33
4.2.Chemokine production of mPSC induced by LPS 34
4.3.The interaction between macrophage and mPSC via paracrine effect 35
4.4.The polarization of macrophages 36
Chapter 5. Conclusion 38
Figures and Tables 41
References 61
 

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