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研究生:林軒
研究生(外文):Hsuan Lin
論文名稱:針對慢性阻塞性肺炎所開發的基因及細胞療法
論文名稱(外文):Gene and cell based therapy for chronic obstructive pulmonary disease
指導教授:吳成文
指導教授(外文):Cheng-Wen Wu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:51
中文關鍵詞:慢性阻塞性肺炎幹細胞前驅細胞基因療法肺泡上皮細胞
外文關鍵詞:COPDLungstem cellprogenitor cellgene therapyalveolar epithelial cell
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自從2014年以來,慢性阻塞性肺炎已經位居全球十大死因的第三位,而且目前並無有效的治療方式。慢性阻塞性肺炎的成因主要是由長期的慢性發炎所造成,對肺泡上皮細胞造成不可逆的傷害,引發肺泡細胞凋亡以及胞外基質喪失。在臨床上,慢性阻塞性肺炎的症狀主要有慢性支氣管炎,氣管的發炎及重塑,以及喪失用於氣體交換的肺泡表面積。在我們目前的研究,我們在老鼠上利用彈力蛋白酶來引發肺氣腫,試圖瞭解及分析肺泡的內生性修復機制,且評估基因及細胞療法對於慢性阻塞性肺炎效用及可行性。我們發現在沒有治療的狀況之下,肺泡上皮細胞確實在24小時內便會因應肺損傷開始進行增生分裂,但這些細胞增生在最後並不足以對肺泡進行有效的修復。非常意外地,我們發現第一型肺泡上皮細胞先於第二型肺泡細胞開始進行分裂。我們在用彈力蛋白酶引發肺損傷的老鼠體內利用PEI將基因Bmi-1和Oct-Sox2-Klf4-cMyc(OSKM)送入肺泡上皮細胞便可有效的增進肺功能,肺泡表面積以及肺泡細胞增生。這些新增生地肺泡上皮細胞通常為第一型及第二型肺泡上皮細胞,且並沒有形成癌細胞。我們將Ocr4+的肺部幹/前驅細胞送入用彈力蛋白酶造成肺損傷的老鼠便可增加細胞的分裂及增生約1.7倍,且這些細胞大多為第一型肺泡上皮細胞。總而言之,我們的研究顯示基因及細胞療法在慢性阻塞性肺炎的治療上有機會成為有效的治療策略。更進一步的,我們希望能找到內生性肺泡再生中關鍵的基因,且利用這些基因設計更有效的治療方式。
Chronic obstructive pulmonary disease (COPD) has became the third leading cause of death worldwide since 2014, and currently still lacks efficient treatment. Pathogenesis of COPD is characterized by the chronic inflammatory injuries that lead to irreversible events in lung epithelia such as apoptosis of epithelial cells and proteolysis of extracellular matrix components. The clinical manifestations of the COPD syndrome include chronic bronchitis, a condition of large-airway inflammation and remodeling, and emphysema, the loss of surface area for gas exchange. In the current study, using mouse model of elastase-induced emphysema, we tried to analyze the endogenous repair mechanism of alveolus, and evaluated the potentials of gene or cell-based therapy for the disease. We found that in the absence of therapeutic treatment, alveolar epithelial cells (AECs) actually started to proliferate within 24 hr in response to injury, but finally were insufficient to mediated an efficient repair of alveoli. Unexpectedly, the type-1 AECs (AECs-1) proliferated prior to AEC-2 after injury. In vivo gene delivery of Bmi-1 alone or Oct4-Sox2-Klf4-cMyc cassette (OSKM) in AECs using PEI after elastase induced injury significantly improved lung function, alveolar surface area, and cell proliferations. The newly proliferated AECs showed normal AEC-1 and -2 phenotype, without symptom of oncogenesis. Transplantation of Oct4+ stem/progenitor cells in mice with elastase injury increased 1.7-fold of proliferated cells, and most of which were AEC1 phenotype. In summary, our study showed that gene or cell based therapy could be promising strategies for the treatment of COPD, which currently has no effective pharmaceuticals. Further insights in endogenous alveolar repair mechanism will assistant in uncovering the genes crucial for alveolar regeneration, and designing more efficient therapeutic strategies.
Abstract - - - - i
中文摘要 - - - - ii
Content - - - - iii
List of figure - - - - vi
List of table - - - - vi
Introduction - - - - 1
1. Chronic Obstructive Pulmonary Disease - - - - 1
1.1 Epidemiology of COPD - - - - 1
1.2 Syndromes and of COPD - - - - 1
1.3 Development of COPD - - - - 2
1.4 Diagnosis and Therapeutic treatment for COPD - - - - 3
2. Alveolar epithelium regeneration: mechanisms and stem cell population - - - - 3
2.1 Structure and cell types of alveoli - - - - 3
2.2 Alveolar epithelium injury, regeneration and putative stem/progenitor cells - - - - 4
2.3 Source of AEC1s - - - - 4
3. Cell therapy for lung diseases and injury - - - - 5
3.1 Mesenchymal stem cell - - - - 5
3.2 Alveolar epithelial cells, iPSCs and lung engineering - - - - 5
4. Gene therapy for lung diseases and injury - - - - 6
4.1 Pros and cons - - - - 6
4.2 Gene delivery and PEI based gene therapy - - - - 6
5. Working hypothesis - - - - 7
Method and material - - - - 8
Animals - - - - 8
Injury(Elastase), BrdU labeling and sacrifice - - - - 8
Immunohistochemistry and immunofluorescence staining of tissue - - - - 8
Quantification of BrdU ratio, surface area, and cell type - - - - 10
Pulmonary stem/progenitor cell isolation and differentiation assay - - - - 10
Immunofluorescence staining of cells 12
Result - - - - 13
AECs proliferated in response to elastase-induced emphysema - - - - 13
Slow cycling AECs proliferated in response to elastase-induced emphysema - - - - 14
PEI stemness gene therapy for COPD - - - - 15
Pulmonary stem/progenitor cells differentiated into AEC1-like cells - - - - 17
Transplantation of pulmonary stem/progenitor cells increased proliferation of AEC - - - - 18
Discussion 19
Stemness gene expression after elastase-induced emphysema - - - - 19
Cell type transition of slow cycling AECs - - - - 21
PEI stemness gene therapy - - - - 21
Cell therapy for COPD - - - - 23
Figure - - - - 24
Supplementary data - - - - 44
Reference - - - - 47

Figure.1 - - - - 24
Figure.2 - - - - 28
Figure.3 - - - - 31
Figure.4 - - - - 38
Figure.5 - - - - 41
Sup.1 - - - - 45
Sup.2 - - - - 45

Table.1 - - - - 44
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