臺灣博碩士論文加值系統

靈芝蛋白(LZ-8)和小孢子靈芝(GMI)分別是從Ganoderma lucidum 和 Ganoderma microsporum菌絲體所萃取出具有免疫調節功能的蛋白質。在先前研究發現在處理rLZ-8和GMI之後會抑制非小細胞肺癌的增生，但是對於rLZ-8和GMI處理之後在細胞內的機轉尚未清楚。此篇論文主要研究在處理rLZ-8和GMI後， rLZ-8和GMI會在細胞內的內質網中堆積，並透過PERK-eIF2-ATF4路徑導致內質網壓力，但不會去誘發內質網壓力所產生的細胞凋亡，進而去探討在處理rLZ-8和GMI之後會有LC3-II表現量上升的現象，代表著有自噬反應的現象發生。我們發現在共同處理免疫調節蛋白以及自噬反應抑制劑，發現會使得非小細胞肺癌的存活率有下降的現象。另外，在處理rLZ-8和GMI後發現會有內皮生長因子受器的內吞作用，並且在免疫螢光染色的實驗中發現內皮生長因子受器與rLZ-8和GMI有高度重疊的現象。總括上述，rLZ-8和GMI直接或是間接地利用上皮生長因子受器進入細胞誘導細胞內質網壓力以及造成自噬反應；未來共同處理免疫調節蛋白以及自噬反應抑制劑可做為另一種對於降低非小細胞肺癌存活率的新治療方式。

Ling-Zhi-8 (LZ-8) and GMI, were cloned and purified from Ganoderma lucidum and Ganoderma microsporum, respectively. LZ-8 and GMI effectively prevent lung cancer cells proliferation and mobility in vitro. In this study, we found that the recombinant LZ-8 (rLZ-8) and GMI induce autophagy and endoplasmic reticulum (ER) stress in human non-small cell lung cancer (NSCLC) cells. We demonstrated that rLZ-8 and GMI increase LC3 conversion in a time-dependent manner. More, we found that rLZ-8 and GMI accumulate in the cytosol and colocalization with the ER, Base on the above results, we found that rLZ-8 and GMI accumulated in the ER and stimulated ER stress-mediated downstream signaling pathway, PERK-eIF2-ATF4 axis, but did not induce ER stress-mediated CHOP/GADD153 promoting apoptosis. Moreover, we found that rLZ-8 and GMI regulated ATG5 and beclin1 expression, resulting to induce autophagy. The combination of FIPs and chloroquine, lysosome inhibitor, significantly decreased viability in NSCLC cells. In other way, we observed that the localization of EGFR had high correlation with rLZ-8 and GMI. In conclusion, we suggested that rLZ-8 and GMI induction of endoplasmic reticulum-stress and autophagy may involve in EGFR. This is the first study to reveal the novel anti-cancer function of rLZ-8 and provide evidence that combination treatment with rLZ-8 and pharmacological autophagy inhibitors will be an effective therapeutic for eliminating NSCLC cells.

Abstract i
中文摘要 ii
Introduction 1
Materials and Methods 5
Cell lines 5
Reagents and antibodies 5
Cell viability assay 6
Confocal microscopy 6
Western blot analysis 6
Design and transfection of shRNAs 7
Statistical analysis 7
Results 8
FIPs inhibit cell proliferation in NSCLCs. 8
FIPs accumulate at ER. 8
FIPs induce ER stress via PERK-eIF2-ATF4 pathway. 9
FIPs induce autophagy in human cancer cells. 9
Effect of autophagy inhibitors on FIPs-mediated autophagy and cell viability. 10
FIPs treatment cause EGFR endocytosis and colocalized with EGFR. 11
Discussion 12
References 15
Figure legends 20
Figure 1. Effect of FIPs on cell viability in lung cancer cells in vitro. 20
Figure 2. Localization of FIPs in lung cancer cells with FIPs treatment. 23
Figure 3. Induction of ER-stress in lung cancer cells by FIPs treated. 30
Figure 4. Induction of autophagy in lung cancer cells by FIPs treatment. 35
Figure 5. Effect of autophagy inhibitors on FIPs-mediated autophagy and cell viability. 40
Figure 6. FIPs treatment cause EGFR endocytosis and colocalized with EGFR. 43
Appendixes 48
Table I. Sequences of MAP1LC3B shRNA 48
Appendix Figure 1. 49
Appendix Figure 2. 50

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