臺灣博碩士論文加值系統

CD44不但是一個常用以篩選癌幹細胞的生物標記，也被證明在維持及增進大腸癌細胞的幹性 (stemness) 上扮演重要角色。本實驗室先前的研究結果顯示，當HCT-15和HCT-116人類大腸癌細胞表面的CD44與玻尿酸 (hyaluronic acid, HA) 結合時，會透過活化c-Src激酶使Snail表現增加並轉移至細胞核內，該轉錄因子可直接抑制miR-203的表現，使癌細胞的幹性增加。
CD44之初級RNA經選擇性剪接 (alternative splicing) 後產生不同的RNA，可被轉譯成CD44標準型 (CD44 standard form, CD44s) 以及不同的CD44變異型 (CD44 variant form, CD44v)；而CD44變異型主要表現在癌組織當中。其中，CD44v6近來被發現在大腸癌幹細胞內，可藉由協助c-Met受器的訊息傳遞，活化上皮-間質轉化 (epithelial-mesenchymal transition, EMT)，而促進細胞之移行，侵犯及惡性轉移。但過去對CD44之研究多偏重於探討其在癌細胞惡質化上扮演之角色，對於CD44變異型與標準型在正常腸道上皮細胞過度表現，能否導致其轉型 (transformation) 則尚未被報導。因此在本研究中，利用已知可被突變的K-Ras基因轉型的IEC-6大鼠小腸上皮細胞為模式，先分析在其內過度表現CD44v6或CD44s，能否造成轉型，進而分析此兩種CD44造成IEC-6細胞轉型的能力是否不同。結果顯示，過度表現CD44v6與CD44s的IEC-6細胞，皆呈現較高的飽和密度以及在無血清培養時有較好的存活率。經檢測兩種細胞株內一些參與調節細胞凋亡相關因子的表現後，發現CD44v6過度表現細胞內的Bcl-2及Bcl-xL蛋白表現量皆上升，而dye exclusion和流式細胞分析的結果也顯示其抗凋亡能力也較好。此外，過度表現CD44v6的細胞株不但在軟洋菜膠形成聚落的能力優於過度表現CD44s的細胞株，其在定義培養液 (defined medium) 形成的球體數目與體積也遠超過後者，其中腸道(癌)幹細胞標記Lgr5蛋白表現也明顯增加。
綜言之，CD44s或CD44v6的過度表現都能造成IEC-6正常腸道上皮細胞的轉型，但CD44v6引發該細胞轉型的能力遠超過CD44s。而CD44v6造成正常腸道上皮細胞轉型的確切機轉，以及其與CD44s之間作用的差異，都值得未來更深入的研究。

CD44 is not only a biomarker frequently used for isolating various CSCs, but also contributes to their stemness. Our previous study has shown that hyaluronic acid (HA), by interacting with CD44, activates c-Src kinase which subsequently upregulates the expression of the Snail which in turn directly diminishes the expression of miR-203, a well-known tumor suppressor and a stemness inhibitor, leading to an increase in the stemness of HCT-15 and HT-29 human CRC cells. Due to an alternative splicing of its primary transcript, CD44 proteins can exist as either a standard (CD44s) or different variant (CD44v) isoforms and the latter ones are predominantly expressed in tumor tissues. Among CD44v’s, the CD44v6 was found to cooperate with c-Met and activate EMT, hence increasing migration, invasion and metastasis of CRC cells.
Even though the crucial roles of CD44, especially its variant isoforms, in the malignant progression of different types of tumors were well documented, little is known about the transformation abilities of CD44s and CD44v in normal intestinal epithelial cells. To answer these intriguing questions, ICE-6 normal rat small intestinal epithelial cells were chosen as a model and stable clones overexpressing human genes encoding CD44v6 and CD44s, respectively, were established. I found that although the growth rates of both CD44v6- and CD44s -overexpressing clones were similar to that of the parental IEC-6 cells, they exhibited higher saturation density and better survival, respectively, in a regular and a serum-free condition. The latter was due to a stronger anti-apoptotic effect. In accordance, protein levels of two anti-apoptotic factor, Bcl-2 and Bcl-xL, were found to be upregulated in CD44v6-overexpressing clones. Moreover, these clones also formed more colonies in soft agar and more large-size spheres in a defined medium than the CD44s-overexpressing clones. Intriguingly, the levels of Lgr5, a marker for both normal and cancerous intestinal stem cells, were markedly increased in CD44v6-overexpressing clones.
Taken together, overexpression of both CD44v6 and CD44s in IEC-6 normal intestinal epithelial cells could induce their transformation, and the former had a much higher potency than the latter. These observations warrant a further investigation of the mechanisms underlying CD44v6-induced transformation of IEC-6 cells as well as that underlying a stronger transforming ability of CD44v6.

總目錄
總目錄.....................................I
圖次目錄...................................II
中文摘要...................................III
英文摘要...................................V
緒論.......................................1
研究動機...................................9
材料方法...................................10
實驗結果...................................16
討論.......................................20
參考文獻...................................23
圖表.......................................29
附錄.......................................39

圖次目錄
Figure 1. CD44v6 and CD44s levels in the CD44v6- and CD44s-overexpressing IEC-6 clones.....................29
Figure 2. Higher saturation densities are observed in CD44v6- and CD44s-overexpressing IEC-6 clones................................................30
Figure 3. The growth rates of the parental IEC-6 cells as well as the CD44v6- and CD44s-overexpressing clones are similar...............................................31
Figure 4. CD44v6- and CD44s-overexpressing IEC-6 clones survive better in a serum-free condition.............................................32
Figure 5. Lower apoptotic populations are found in CD44v6-1, v6-2 and CD44s-1 IEC-6 cells cultured under a serum-free condition.............................................33
Figure 6. Expression levels of several anti- and pro-apoptotic factors in CD44v6- and CD44s-overexpressing IEC-6 clones..........................................34
Figure 7. Higher soft agar colony-forming abilities are found in the CD44v6-1, v6-2 as well as CD44s-1 IEC-6 clones................................................35
Figure 8. CD44v6-overexpressing IEC-6 clones have higher sphere-forming abilities.............................................36
Figure 9. The expression levels of several stem cell markers in the CD44v6- and CD44s-overexpressing IEC-6 clones................................................37
Table 1. The comparison between CD44v6- and CD44s-overexpressing clones in several assays................................................38

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