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研究生:呂政鴻
研究生(外文):Cheng-Hung Lu
論文名稱:絲胺酸蛋白酶抑制劑基因SERPINB13對攝護腺癌細胞之影響
論文名稱(外文):The Cellular Effects of SERPINB13 in Prostate Cancer Cells.
指導教授:徐邦達徐邦達引用關係
指導教授(外文):Ban-Dar Hsu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:58
中文關鍵詞:絲胺酸蛋白酶抑制劑攝護腺癌SERPINB13PC-3癌轉移活體外癌細胞侵襲實驗
外文關鍵詞:Serine proteinase inhibitorprostate cancermetastasisin vitro invasion assay
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過去十年來在國人罹患攝護腺癌 (prostate cancer) 的比例與死亡率逐年增加,目前已高居台灣地區第八大癌症死因,攝護腺癌早期發現,其存活率可高達 90% 以上,但若發生癌轉移則存活率將降至30%左右,如能有效抑制攝護腺癌的侵襲與轉移,將有助於罹癌病患存活率的提升。骨頭是攝護腺癌中僅次於淋巴結最常發生轉移的部位,攝護腺癌會加快骨生成與骨喪失作用,骨喪失作用的增加會使細胞外間質釋放某些因子促使癌生成,蛋白酶Cathepsin K在骨蝕作用中會分解細胞外間質,而已被證實是絲胺酸蛋白酶抑制劑SERPINB13 的目標蛋白酶,而絲胺酸蛋白酶抑制劑SERPINB13基因是SerpinB 家族中的新成員,該家族中有十個成員均位於人類染色體18q21.3的位置,與它相鄰的SERPINB5已被證明是抑癌基因。再者第十八條染色體發生異質性消失的情形與許多癌症具有關連性,基於相同基因cluster的基因組可能具有相似的功能,所以推測SERPINB13在攝護腺癌可能具有類似的功能。SERPINB13基因具有三種不同的截接型態,其差異發生在位於helix C與helix D之間的interhelical loop,依據interhelical loop 長度的減少我們將它們稱之為SERPINB13(0)、SERPINB13(-9)、SERPINB13(-10)。
為了探討SERPINB13與攝護腺癌的關係,本實驗先以反轉錄聚合酵素連鎖反應RT-PCR分析此基因在攝護腺中的表現情形,結果顯示SERPINB13僅表現在正常的攝護腺細胞株PZ-HPV-7中,在良性CA-HPV-10 攝護腺癌細胞株中表現略減,而在已發生轉移的攝護腺癌細胞株LNCap與PC-3中則不表現,呈負調控。接著活體外癌細胞侵襲技術篩選較具癌侵襲能力之細胞株,由於 PC-3明顯較 LNCaP 細胞株更具侵襲轉移能力,因此本論文選擇 PC-3 惡性攝護腺癌細胞株作為後續活體外癌侵襲模擬實驗的研究材料。首先建構含這三種不同截接型態的SERPINB13表現質體,將之轉殖入PC-3細胞株中後,以西方墨點法進一步確認PC-3攝護腺癌細胞內,三種不同截接型態之等融合蛋白皆被成功表現,接著利用細胞增生實驗確認不同截接型態的 SERPINB13 並不會影響攝護腺癌細胞的增生能力後。取轉殖的細胞株做活體外癌細胞侵襲實驗,結果發現當PC-3 攝護腺癌細胞大量表現SERPINB13時,其癌侵襲能力遠較僅轉殖 EGFP 對照組質體的 PC-3 細胞要來得高許多,但對於促進PC-3攝護腺癌細胞侵襲能力的影響,卻會隨著interhelical loop長度的增加而有減緩之趨勢。
本篇論文顯示SERPINB13基因在攝護腺癌大量表現時會促進細胞的侵襲轉移能力,可證明它與攝護腺癌具有相關性,雖然詳細的作用機制並不清楚,但可提供我們在防止攝護腺癌轉移上一個新的思考方向。
The incidence rate and death rate of prostate cancer have been rapidly increasing in the past 10 years in Taiwan. It has became the eighth most common cause of cancer-related death. The survival rate for patients diagnosed with prostate cancer at early stage is 90%. As it happens to metastasis, the survival rate drops to 30%. Preventing and inhibiting the further invasion and metastasis of prostate cancer will raise the survival rate for patients who suffer from prostate cancer. Bone is second most common site of metastasis (after lymph nodes) for the prostate cancer. Lesions associated with prostate cancer generally exhibit increased bone formation and resorption. Increased bone resportion may release factors from the extracellular matrix that contribute to tumor growth. Cathepsin K is a protease that exhibits strong degradative activity against the extracellular matrix. It is the target protease of SERPINB13.
Serine proteinase inhibitor SERPINB13 has been grouped into the cluster of clade B serpins located at chromosome 18q21.3. SERPINB5 was a tumor suppressor gene in the same cluster This region is a known area for lost of heterozygosity (LOH) and associated with many malignancies. On the basis of this reason, SERPINB13 may represent an equivalent gene to SERPINB5 in prostate cancer. SERPINB13 gene has three different splicing forms. They show altered lengths of the part of exon 3 that forms interhelical loop, According to the decreasing of the interhelical loop,we call them SERPINB13(0), SERPINB13(-9) and SERPINB13(-10).
To know the role of SERPINB13 in prostate cancer, we examined the SERPINB13 expression in prostate cell lines by RT-PCR. The expression pattern illustrates SERPINB13 downregulation in prostate cancer . Then screen a more invasive prostate cancer cell line PC-3 as our candidate cell line by in vitro invasion assay. Three constructed plasmids with different splicing forms were transfected into PC-3 cells. First of all, the western blot confirmed the expression of SERPINB13 and the growth rate of PC-3 cells was not changed by transfection in the cell proliferation test. In the in vitro invasion assay of SERPINB13 transfected PC-3 cells, PC-3 cells overexpress SERPINB13 were more invasive than vector alone. The ability of invasion was decreasing as the increasing of the interhelical loop.
The results show prostate cancer overexpresses SERPINB13 will increase invasion and metastasis ability. It can be proven to be related to prostate cancer. Understanding the detailed mechanism of SERPINB13 remains an ongoing challenge. But it can provide us a new aspect in protecting prostate cancer metastasis.
一、 中文摘要………………………………… 1
二、 英文摘要………………………………… 3
三、 前言……………………………………… 5
四、 材料與方法……………………………… 14
五、 結果……………………………………… 28
六、 討論……………………………………… 33
七、 參考文獻………………………………… 37
八、 圖表……………………………………… 43
九、 附錄……………………………………… 60
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