大多數的人類肺腺癌被發現生長於肺部的周邊，推測這些腫瘤可能是肺泡或末端細支氣管遠端的肺上皮細胞轉型所引發。先前的研究發現，位於細支氣管肺泡管結（BADJ）的細支氣管肺泡幹細胞（BASC），其對『萘』具抗性，具有自我更新的特性；並可同時表達Club細胞和II型肺泡細胞的標誌物CCSP和pro-SPC蛋白。在細胞培養和肺腫瘤小鼠模型中，發現BASCs可因致癌K-Ras刺激而增生，推測致癌基因突變的BASCs可能是肺腺癌的前體細胞。然而，尚未有直接證據。因此，我們利用CCSP-CreER / LSL-LacZ / CCSP-rtTA / tetO-EGFRL858R和CCSP-CreER / LSL-LacZ基因工程小鼠，先以遺傳標記方式追蹤抗萘損傷的細支氣管上皮細胞，如BASCs和variant club cells，接著再觸發致癌EGFR的表現，同樣地，我們也以CCSP-CreER /Brainbow2.1 / CCSP-rtTA / tetO-EGFRL858R小鼠模型進行『萘』抗性細胞的譜系追踪，結果亦顯示這些具有萘抗性細胞可被轉化後發展成腫瘤，且腫瘤位置於周邊肺臟，證明致癌突變的BASC可以是肺腺癌的起源細胞。特別的是，由萘抗性發育而來的腫瘤之增生速率比未經『萘』處理的控制組慢，且腫瘤表達更高量的肺腺癌標誌物TTF-1。另一方面，在末端細支氣管中發現具有較多的螢光標記腫瘤在萘受損肺組中，符合在臨床上肺腺癌起始於末端的現象。我們於正常人類肺臟支氣管上皮細胞NL-20表達EGFRL858R，並以懸浮培養，發現可促進CCSP/proSPC雙陽性的細胞群，並且細胞有較高的幹細胞特性蛋白 Sox9的表現。總結，我們的結果顯示，BASCs的致癌EGFR突變可造成肺遠端肺腺癌的形成。

Most lung adenocarcinomas located peripherally at the lungs, suggesting these tumors initiated from the cells niched at distal lung epithelium of alveoli or terminal bronchioles. Previous research showed that bronchioloalveolar stem cell (BASC) locates at bronchioalveolar duct junction (BADJ). BASC expresses both CCSP and proSPC proteins that are markers of Club cells and type II pneumocytes, respectively. BASC is resistant to naphthalene toxicity and displays self-renewal characters as stem cells. The number of BASC was increased in both culture cells and lung tumor model in response to oncogenic K-Ras stimuli. However, whether oncogene leads to BASC transformation into adenocarcinoma remains unclear. We thus genetically labeled the remaining cells of naphthalene-injured airway epithelium, presumably BASC and variant club cells, followed by triggering oncogenic EGFR stimuli in CCSP-CreER/LSL-LacZ/CCSP-rtTA/tetO-EGFRL858R and CCSP-CreER/LSL-Brainbow2.1/CCSP-rtTA/tetO-EGFRL858R mouse models. The lineage tracing result showed these naphthalene-resistant cells were transformed and later developed into lung tumors, indicating oncogenic mutation in BASC could be the origin of lung adenocarcinoma.　The cells in lung tumors cells of naphthalene-injured lungs displayed a lower proliferation rate than tumor cells in the uninjured lungs, while expressed higher levels of lung adenocarcinoma marker TTF-1. In addition, most Brainbow labeled tumors in the naphthalene-injured lungs were found in terminal bronchioles, consistent with the clinical observation that lung adenocarcinomas initiate at the peripheral lung. Likewise, an increased population of CCSP and proSPC dual positive BASC-like cells was detected in EGFRL858R expressing human lung epithelial NL-20 cell stable clone in sphere cell culture. These BASC-like cells also showed an increased expression level of stem cell marker Sox9. Altogether, our findings revealed that oncogenic mutation in distal lung BASC contributes to lung adenocarcinoma.

中文摘要 1
Abstract 2
Contents 4
Introduction 7
Lung adenocarcinoma 7
Causes of lung cancer 8
Distal lung epithelial cells 8
Naphthalene-resistant cells 10
Relationship between lung adenocarcinoma and lung epithelial cells 10
Tumor lineage tracing principle 11
EGFR pathway and mutation 12
Hypothesis 14
Result 15
CCSP-CreER/LSL-LacZ/CCSP-rtTA/tetO-EGFRL858R mouse model is generated for tumor lineage tracing 15
Naphthalene-resistant cells are tumor-initiating cells 16
BASC is one of the important tumor-initiating cells found in CZE mice 16
Identifying individual tumors by lineage tracing with multiple-fluorescence labeling 17
BASCs have a higher incidence to form tumors than other progenitor cells 18
The size of tumor formed by BASCs is relatively small 19
BASCs contribute to terminal bronchioles tumor formation 19
Expression of BASC-related genes, SPC and Sox9 were increased in early-stage lung tumor of the post-injury mouse 20
Establish inducible construction of EGFRL858R expression on NL-20 cells 21
EGFR mutation slightly stimulate spheroid formation 22
Expression of EGFRL858R in NL-20 sphere cells promotes CCSP/SPC positive cell expansion 22
Activated EGFR pathway enhances Sox9 and BASC-related genes expression 23
Discussion 25
Figure 29
Figure 1. The CZE mouse model mechanism and processing is generated for tumor lineage tracing 29
Figure 2. Naphthalene-resistant cells repair bronchioles at BADJ regions and also cause tumor formation 31
Figure 3. Different tumors were found in CCSP promoter driven oncogene mice, tumors from BASC is accounts for about 20% 33
Figure 4. Brainbow2.1 system can label tumors with different fluorescence to distinguish tumor distribution 35
Figure 5. More tumors were present in the post-injury group. 36
Figure 6. The area of tumor formed by BASCs is relatively small 37
Figure 7. Naphthalene-treated mice exhibits higher lung adenocarcinoma marker 38
Figure 8. BASCs contribute to terminal bronchioles tumor formation 39
Figure 9. Expression of BASC-related genes, SPC and Sox9 were increased on 2, 3 months post-injury mouse. 40
Figure 10. Doxycycline-inducible expression of EGFRL858R perform in NL-20 41
Figure 11. The p-AKT and p-Erk1/2 was elevated by EGFRL858R expression 42
Figure 12. The NL-20 cell sphere culture to enrich lung stem cell 43
Figure 13. Expression of EGFRL858R in NL-20 sphere culture slightly increases spheroid size. 44
Figure 15. BASC-related genes were up-regulate by expression of EFGRL858R 46
Methods 47
Mouse 47
Tamoxifen, doxycycline and naphthalene administration 47
X-gal staining 48
Fluorescent sample processing 48
Fluorescent sample analysis 49
Immunofluorescence (IF) 49
Immunochemistry (IHC) 50
Cell culture 50
Plasmid establishment 51
Lent-viral transduction 52
Sphere culture 52
Flow cytometry 53
Western blot 53
RNA isolation and Quantitative qPCR 54
Statistical analysis 55
Appendix 56
Supplementary figure.1 Tumor without β-galactosidase expression was found in CZE mice. 56
Supplementary figure.2 Population of Ki-67 and TTF-1 positive cell in CBE mice 57
Supplementary figure.3 pINDUCER20-mCherry-EGFRL858R plasmid establishment process 59
Supplementary figure.4 compensation of flow cytometry analysis 60
Table.1 list of primer for construct 61
Table.2 list of primer for qPCR 61
Table.3 list of antibody 62
Reference 63

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