化療藥物的副作用和癌症細胞的抗藥性是造成其現今仍無法非常有效治療癌症的重要因素，這也是為什麼找出可以增加癌症細胞對化療藥物之敏感性的方法和藥物是現在一個熱門的議題。此外，癌症幹細胞也被認為是造成癌症細胞抗藥性的一個重要的因子；在癌症幹細胞的理論中認為，癌症幹細胞不像一般的癌症細胞，它不但對化療藥物具有很大的抗性，並且它可以分化成不同型態的癌症細胞，使將被清除的腫瘤產生復發或產生更強的抗藥性。在本篇研究中，我們將探討合併使用傳統中草藥靈芝多醣體和臨床化療藥物順鉑在A549非小細胞肺癌細胞株(A549)和A2臨床肺癌幹細胞(A2)中的療效。我們的研究顯示，比起投以任何單獨一種藥物，同時投以靈芝多醣體和臨床化療藥物順鉑(一種能與DNA進行鍵結，而使細胞無法正常分裂，最終導致細胞死亡的藥物)能夠更有效的殺死A549和A2細胞。並且在合併使用靈芝多醣體和順鉑的狀況下，會誘導A549產生”自體吞噬”的現象，進而殺死更多的A549細胞。接下來，我們發現誘導自體吞噬的關鍵角色的蛋白質-Akt，在合併投藥的情況下，有大量減少的情形。另一方面，相比投以任何單一藥物，合併投藥同樣也可以使A2細胞產生更多細胞死亡的現象；不同的是，合併投藥誘導A2細胞產生更多的細胞凋亡，而並非是透過自體吞噬讓細胞死亡。而接著我們發現，靈芝多醣體是透過減低A2癌症幹細胞的幹細胞特性，而使本身的抗性降低，導致順鉑能更有效的使A2細胞產生細胞凋亡。總體來說，我們的研究認為靈芝多醣體和臨床化療藥物順鉑合併用藥的治療方式也許可以成為一個很有效的策略來改善現在化療藥物所面臨的抗藥性以及副作用所帶來的限制。

ide-effect and resistance of chemotherapeutic reagent are crucial factors that limited its usage. That why sensitization of cancer cell to chemotherapeutic reagents is a hot issue now. Besides, cancer stem cell is considered an important factor in drug-resistant. The cancer stem cell hypothesis suggests that, unlike most cancer cells within a tumor, cancer stem cells resist chemotherapeutic drugs and can regenerate the various cell types in the tumor, thereby causing relapse of the tumor or dosing mass anti-cancer drug. In our study, we explored the combined effects of combining traditional Chinese medicine, extract of Reishi polysaccharides (EORP) and chemotherapeutic reagents cisplatin on the elimination of A549 lung cancer cell line and primary cancer stem-like cells. Our results showed that combining EORP and a platinum-containing anti-cancer drug cisplatin, which causing crosslinking of DNA, and triggers apoptosis, was more effective in killing A549 human lung cancer cell lines and A2 primary lung cancer stem-like cells than either EORP or cisplatin single. The combination of EORP and cisplatin was synergistic induced autophagy, which also called type II programmed cell death, causing more cell death than any single treatment in A549. Furthermore, we also found that the expression of Akt, the key protein in induction of autophagy, was significantly synergistic reduced by the combined treatment. Moreover, similarly effect was observed in A2. The combined treatment was more effectively killing A2 than either single treatment. But unlike induction of autophagy in A549, the combination of EORP and cisplatin through reduced properties of cancer stem cell in A2 lead to cell apoptosis. Taken together, the present study reveals that a combination of EORP and cisplatin may be a effectively strategy for improving the problems of side-effect and resistance of chemotherapeutic reagents.

Content
中文摘要 3
Abstract 5
Introduction 7
Material and Methods 12
Preparation of EORP 12
Cell culture 12
MTT assay 12
Median-Effect Analysis of Combined Effects 13
Preparation of total cell lysate 13
Western Blotting Analysis 13
Cell cycle analysis 14
Apoptosis analysis (Vybrant ® Apoptosis Assay Kit) 14
Spheres Culture 15
Result 16
Cytotoxicity of EORP in A549 human lung cancer Cell line 16
A549 human lung cancer cell line was sensitized to anti-cancer drug cisplatin by EORP 16
Cisplatin combined treatment with EORP increased cell death phenomenon caused by cisplatin in A549 human lung cancer cell line 17
A2 human primary lung cancer cells exhibits cancer stem-like properties 18
A2 human primary lung cancer stem-like cell was also sensitized to anti-cancer drug Cisplatin by EORP 18
Cisplatin combined treatment with EORP increased cell death phenomenon caused by cisplatin in A2 human primary lung cancer stem-like cells 19
EORP induced autophagy in A549 human lung cancer cell line but not in A2 human primary lung cancer stem-like cells 20
EORP induced autophagy probably through decreased phosphorylation of Akt in A549 human lung cancer cell line 20
EORP reduce expression level of oct4, c-Myc, and klf4 in A2 human primary lung cancer stem-like cells 21
EORP dose-dependently reduced number of spheres formed by A2 human primary lung cancer stem-like cells 21
Discussion 23
References 29
Figure legend 34

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