臺灣博碩士論文加值系統

在婦科的癌症當中，預後相當不良且最致命的癌症即為卵巢癌，其主要細胞來
源約莫九成由上皮組織而來。而不像其他癌症細胞會藉由入侵血管轉移至遠端，脫
落卵巢癌細胞可直接藉由腹腔內的生理液傳播至腹腔各處。在卵巢腫瘤早期發展過
程當中，許多分子表現量的增減扮演相當重要的角色，這些分子不但可幫助腫瘤細
胞抵抗因無法接觸細胞外基質而進行的失巢凋亡(anoikis)，並且能導致和細胞存活及
增生相關之 AKT分子磷酸化。目前已知 CD164分子和前列腺癌之轉移相關，我們
假設 CD164這個唾液酸黏蛋白分子會參與腫瘤生成(tumorigenesis)，甚至造成癌細胞
的轉移。而且目前由於已知 CD164 在造血前趨細胞中，可因 CXCL12 之刺激與
CXCR4 形成複合物而影響細胞移動的能力。加上已有研究指出 CXCL12 以及它的
受器會參與許多癌細胞的增生及轉移過程。因此我們想探討 CD164分子是否
在腫瘤生成的過程中扮演重要的角色。
實驗結果中發現，CD164 在卵巢上皮癌之細胞株中表現量相較於正常卵巢上皮
細胞會增加許多，而且於卵巢正常上皮細胞株 OSE10中大量表現 CD164分子後，
不僅在細胞型態上發生極大改變，比起野生型及控制組的細胞株而言，引起
Anchorage-Independent Colony Formation 現象也更加明顯。而在惡性邊緣腺瘤細胞
株 ML-46 及正常肺上皮細胞株 BEAS-2B 中也發現相同情形。接著在動物實驗當
中，也證實大量表現 CD164 分子的卵巢上皮細胞株不但可形成腫瘤，而且生長速
度比癌細胞還快上許多。除此之外，當大量表現 CD164分子後，會造成細胞與細胞
間質的黏附能力下降，而細胞之 E-cadherin 及 p-Akt 的表現量會增加，當利用
CXCR4 之抑制劑及 PI3K 抑制劑作用後發現 p-Akt 表現會遭抑制。由於實驗過程
中發現 CD164 分子會導致細胞迅速增生，因此利用流式細胞儀檢測大量表現
CD164 分子之細胞株後，發現其細胞週期的確受到影響。由以上研究結果，強烈暗
示著 CD164分子參與調控卵巢腫瘤生成機制。之後，我們希望能更深入釐清 CD164
在不同癌症生成可扮演之角色，期許在未來可做為臨床上癌症治療的標的之一。

Among the gynecological tumors, the most lethal cancer is the ovarian cancer and about ninety percent of the ovarian cancers are from the epithelial tissue. In contrast to most other type of carcinomas, dissemination of ovarian cancers through vasculature is rare and exfoliation is the most common pathway for metastasis. Exfoliated tumor cells of ovarian cancers are transported by peritoneal fluid and implant on the peritoneum and mesothelial linings of pelvic and abdominal organs. The fluctuating expression of different molecules may play an important role in the processes of ovarian tumor development. These molecules not only help tumor cells to resist anoikis,but can lead to cell survival and proliferation due to activating AKT phosphorylation. Owing to evidences indicating that CD164 might play a role in metastasis of prostate cancer and CD164 can modulate the CXCL12-mediated migration by forming complex with CXCR4 in hematopoietic progenitor cells, we propose that the CD164, a sialomucin, may be involved in tumorigenesis and cancer metastasis. From the preliminary results we found that the expression of CD164 in ovarian epithelial cancer cell lines was higher than normal ovarian epithelial cells and depended on their malignancy. In OSE10, a normal epithelial ovarian cell line, overexpressing CD164 not only changed the cell morphology, but showed more colonies in anchorage-independent colony formation assay than wild-type and vehicle control cells, and the similar results were noted in human ovarian epithelial borderline cell lines, ML46 and in BEAS-2B, a lung normal epithelial cell line using the same assay. Then in the animal model we also found that CD164 overexpressing ovarian epithelial cells not only formed tumors, but grew faster than ovarian cancer cells. Furthermore, overexpression of CD164 caused up-regulation of E-cadherin and p-Akt but suppressed by CXCR4 inhibitor and PI3K inhibitor and the cell cycle was indeed affected. These results strongly suggest that CD164 is involved in the regulation of molecular mechanisms of ovarian tumor formation.

壹 、 中文摘要 ………………………………………….…………………I
貳 、 英文摘要……………………………………………………………...II
參 、 緒論
一 . 腫瘤生成 …………………………………………………………………….1
二 . 卵巢上皮細胞癌(Epithelial ovarian cancer) ………………………………2
三 . CD164 分子 …………………………………………………………………5
四 . CXCL12/CXCR4 axis ………………………………………………………6
五 . 細胞週期……………………………………………………………………...8
六 . 研究動機與方向…………………………………………………………….10
肆 、 實驗方法與材料
一 . 細胞培養 ( cell culture )……………………………………………………12
二 . 質體的抽取與製備………………………………………………….………12
三. 細胞轉染( Transfection )及慢病毒製備 (Lentivirus production)…………...14
四 . 慢病毒感染 ( Lentivirus infection )………………………………………...15
五 . 藥物篩選持續穩定表現之細胞株 ( Stable clone )………………..……….15
六 . 細胞蛋白質萃取 ( Protein extraction )……………………………………..15
七 . 核蛋白萃取………………………………………………………………….16
八 . 西方墨點法 ( Western blotting )……………………………………………16
九 . 細胞週期分析 (Cell cycle analysis)………………………………………..18
十 . Colony formation assay ……………………………………………………...18
十一 .Xenograft Tumors in Nude Mice ………………………………………….19
十二 . 細胞貼附力分析 (Cell adhesion assay)…………………………………19
十三 . 細胞增生試驗 (Cell proliferation assay)………………………………..20
十四 . 利用流式細胞儀分析不同細胞株細胞中CD164 分子之表現 ……….20
十五 . 製備勝任細胞(Competent cells)………………………………………... 21
十六 .統計方法………………………..................................................................22
伍 、 結果
一 . 確認不同細胞株之CD164 表現量…………………………………………23
二 . 藥物篩選過度表現CD164 基因之效率……………………………………23
三 . 使用流式細胞儀探討不同細胞株中CD164 分子之表現量………………23
四 . 野生型、控制組及過度表現CD164 之OSE10 及BEAS-2B 細胞之型態…24
五 . 探討CD164 分子對於細胞貼附力影響分析………………………………24
六 . 探討CD164 對於上皮-間質轉化相關分子之影響………………………..25
七 . 探討CD164 對於細胞核內EMT 相關分子之影響………………………..25
八 . 探討EMT 相關分子在不同細胞株之表現量……………………………..26
九 . 探討CD164 對於細胞增生速率之影響……………………………………26
十 . 探討CD164 分子對於腫瘤生成(tumorigenesis)之影響…………………..27
十一 . Xenograft Tumors in Nude Mice …………………………………………27
十二 . CD164 分子對細胞週期之影響 …………………………………………28
十三 . 探討CD164 分子對於AKT 磷酸化,BAX 及Bcl-2 之影響…………… 29
十四 . SDF-1以及CXCR4 抑制劑AMD3100 對於大量表現CD164 之OSE10
細胞株及控制組(Vehicle)細胞株訊息傳遞之影響………….………..29
十五 . 探討存在 SDF-1刺激之下，AMD3100 對於大量表現CD164 之OSE10
細胞株及控制組(Vehicle)細胞株訊息傳遞之影響………….…………..30
十六 . 探討PI3K 抑制劑LY294002 對於大量表現CD164 之OSE10 細胞株及
控制組(Vehicle)細胞株訊息傳遞之影響………………………………31
十七 . 利用AMD3100 及LY294002 探討其對於過度表現CD164 分子之OSE10
細胞株之Anchorage-Independent Colony Formation 影響……………31
陸 、 討論 ………………………………………………………………… 33
柒 、 圖表
圖1. CD164 分子在不同細胞株之表現量…………………………………….…39
圖2. OSE10 細胞CD164 基因過度表現之效率………………………………...40
圖3. OSE10 細胞CD164 基因過度表現之效率………………………………...41
圖4 . 野生型、控制組及過度表現CD164 之OSE10 及BEAS-2B 細胞之型態.. 42
圖5 . CD164 分子對於細胞貼附力影響分析………………………..………….43
圖6 . CD164 對於EMT 相關分子之影響……………………………………....44
圖7 . CD164 對於細胞核內EMT 相關分子之影響 ……………………….….45
圖8 . EMT 相關分子在不同細胞株之表現量………………………………… 46
圖9 . CD164 對於細胞增生速率之影響 ……………………………………....47
圖10 . Soft agar colony formation assay…………………………………… ...... 48
圖11 .Xenograft Tumors in Nude Mice ……………………………………..…..49
圖12 .CD164 分子對細胞週期之影響………………………………………….50
圖13 . 探討CD164 分子對於AKT 磷酸化,BAX 及Bcl-2 之影響 ………….51
圖14 . SDF-1以及CXCR4 抑制劑AMD3100 對於大量表現CD164 之OSE10
細胞株及控制組(Vehicle)細胞株訊息傳遞之影響…………………..52
圖15 . 探討存在 SDF-1刺激之下，AMD3100 對於大量表現CD164 之OSE10
細胞株及控制組(Vehicle)細胞株訊息傳遞之影響……………………53
圖16 . 探討PI3K 抑制劑LY294002 對於大量表現CD164 之OSE10 細胞株及
控制組(Vehicle)細胞株訊息傳遞之影響…………………….……..…54
圖17 . 利用AMD3100 及LY294002 探討其對於過度表現CD164 分子之OSE10
細胞株之Anchorage-Independent Colony Formation 影響……..…… 55
圖18 . CD164 分子影響卵巢上皮細胞腫瘤生成模擬圖 ……………………..56
捌 、 參考文獻…………………………………………………………… 57

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