Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20069
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dc.contributor.advisor陳健尉zh_TW
dc.contributor.advisorJeremy J.W. Chenen_US
dc.contributor.author莊雅玲zh_TW
dc.contributor.authorAngelaen_US
dc.date2005zh_TW
dc.date.accessioned2014-06-06T07:11:42Z-
dc.date.available2014-06-06T07:11:42Z-
dc.identifier.urihttp://hdl.handle.net/11455/20069-
dc.description.abstract近年來,臺灣的肺癌病例有顯著增加的趨勢,在女性的癌症病例中因肺癌而死亡的人數已高居首位。癌轉移為一個相當複雜的過程,包含附著、遷移及侵入,並破壞基底膜侵入周圍組織。在文獻中發現,許多的分子會參與細胞的轉移及侵入如 CD44、MMPs、TIMPs 及 E-cadherin,然而癌轉移的分子機制仍然不清楚。就目前所知,癌轉移需要促進轉移基因的表現,並降低抑制轉移基因的表現,因此首先要定義出哪些基因為癌細胞轉移及侵入相關的基因。微陣列 (microarray) 為一種可以大量平行篩檢分析基因表現的有力工具,在先前的研究中,利用侵入/轉移模式的細胞株 (CL1-0,CL1-1,CL1-5) 以及微陣列技術發現 HLJ1 (Human Liver DnaJ-like protein) 是一新的抑癌基因,具有抑制腫瘤細胞轉移與生長的能力。HLJ-1 為 DnaJ-like 熱休克蛋白 (Heat Shock Protein, HSP),屬於 Hsp40 family 中的一員。為研究 HLJ1 於肺腺癌中扮演的角色,於本研究中,將以 RNA 干擾的方式轉染肺腺癌 (CL1-0) 細胞,試圖篩選出 HLJ1 靜默的細胞株,並做後續的功能分析。此細胞株的建立將有助於進ㄧ歩探討 HLJ1 在細胞內所扮演的角色,並探討其調控機轉,找出可能受到 HLJ1 影響的蛋白質。本研究利用即時定量聚合酶連鎖反應與西方墨點法,目前已經篩選到4株能夠穩定抑制 HLJ1 的細胞株,並做後續的功能分析。在功能分析方面將分成五個部分,分別是細胞增生分析、試管內侵入能力分析、細胞遷移及柔軟瓊脂中細胞聚落形成分析,以觀察細胞生物特性情形。結果發現 HLJ1 表現量低的 CL1-0 細胞會增加侵襲、轉移能力及細胞的非依賴固著的能力。此外為了更進一歩探討 HLJ1 在肺癌轉移及侵入過程中可能的調控機轉,所以利用即時定量聚合酶連鎖反應與西方墨點法觀察細胞內蛋白質的變化,發現在低侵襲力的肺腺癌細胞中 (CL1-0) 以 RNAi 干擾技術降低 HLJ1 的表現後,與 Scramble control (Mmix、MI-6、MII-3、MV-5) 及 CL1-0 對照組相比較,結果顯示磷酸化的 ERK 增加,而 E-cadherin 表現降低。因此推論 HLJ1 藉由調節 E-cadherin 表現,進而達到抑制肺癌細胞的侵襲能力。zh_TW
dc.description.abstractRecently, lung cancer has dominantly increased and ranked up to the first place among Taiwanese women died from cancers. Metastasis is a very complex process that involves cell adhesion, migration, invasion and degradation of the surrounding extracellular matrix. Many studies on cancer metastasis have been conducted, and several molecules participating in tumor cell invasion and metastasis have been identified, such as CD44, MMPs, TIMPs, and E-cadherin. However, molecular aspects of metastasis are not clearly understood. To obtain metastatic ability, tumor cells require the coordinated expression of metastasis-promoting genes and down-requlation of metastasis suppressing genes. It is necessary to identify the possible genes associated with cancer invasion and metastasis. Microarray is a powerful tool for screening a large number of genes expressions. By using an invasion/metastasis cell line model and microarray in a previous study, we have identified a tumor suppressor candidate, Human Liver DnaJ-like heat shock protein (HLJ1), which can suppress cancer cell invasion and growth. It is a member of the 40-kDa-heat shock protein family. To identify the role of HLJ1 in lung adenocarcinoma cells, CL1-0 cells were transfected by RNA interference and silenced cell lines were selected in this study. The establishment of HLJ1-silenced cell lines may assist to further investigate functional characterization and molecular mechanism of HLJ1 subsequently. In addition, we want to find out proteins which were influenced by HLJ1. We have selected four cell lines that HLJ1 expression was stably suppressed. We used MTT, invasion, migration and colony formation in soft agarose analysis to determine the cellular functional characterization. The results showed that the reduced expression of HLJ1 could promote cell invasion, migration and anchorage independent growth. To investigate the mechanism of HLJ1 during the process of lung cancer cell migration and invasion, we performed the real-time RT-PCR and Western blotting analyses. The HLJ1-silenced CL1-0 cells could increase the phosphorylation of ERK as compared with scramble and parental CL1-0 controls, while E-cadherine expression is decreased. These findings suggest that HLJ1 can suppress cancer cell invasion ability via regulation of E-cadherine expression.en_US
dc.description.tableofcontents縮寫字對照表 …………………………………………………………… i 中文摘要 ………………………………………………………………… iii 英文摘要 ………………………………………………………………… v 前言 ……………………………………………………………………… 1 第一章 研究目的 ……………………………………………………… 11 第二章 材料與方法 …………………………………………………… 12 一、HLJ1 siRNA誘發表現系統之建立 ……………………………… 12 二、HLJ1 siRNA持續表現系統之建立 ……………………………… 16 三、細胞培養與分盤 …………………………………………………… 21 四、細胞轉染作用(Transfection) ……………………………………… 22 五、經由G418 Sulfate篩選穩定表現短小干擾核醣核酸的細胞株 … 23 六、細胞total RNA萃取 ………………………………………………… 24 七、cDNA的合成 ……………………………………………………… 25 八、蛋白質樣品製備及定量分析 ……………………………………… 27 九、西方墨點法(Western blot) ………………………………………… 27 十、細胞基質侵襲力分析(matrigel invasion assay) ………………… 29 十一、細胞遷移分析(cell migration assay) …………………………… 30 十二、軟瓊脂細胞聚落形成分析 ……………………………………… 30 十三、細胞增生分析 …………………………………………………… 31 十四、Zymography …………………………………………………… 31 第三章實驗結果 ………………………………………………………… 33 第四章討論 ……………………………………………………………… 39 第五章結論 ……………………………………………………………… 46 参考文獻 ………………………………………………………………… 47 圖表 ……………………………………………………………………… 55 附錄 ……………………………………………………………………… 75zh_TW
dc.language.isoen_USzh_TW
dc.publisher生物醫學研究所zh_TW
dc.subjectE-cadherineen_US
dc.subject西方墨點法zh_TW
dc.subjectsilenceden_US
dc.subjectinvasionen_US
dc.subjectHLJ1en_US
dc.subjectERKen_US
dc.subject即時定量聚合酶連鎖反應zh_TW
dc.subject核醣核酸干擾技術zh_TW
dc.subject細胞增生分析zh_TW
dc.subject細胞遷移zh_TW
dc.subject轉移能力zh_TW
dc.subject非依賴固著的能力zh_TW
dc.subject肺腺癌zh_TW
dc.title以核醣核酸干擾技術研究新的腫瘤/轉移抑制基因HLJ1的功能與特性zh_TW
dc.titleFunctional characterization of a novel tumor/metastasis suppressor HLJ1 by RNA interferenceen_US
dc.typeThesis and Dissertationzh_TW
item.cerifentitytypePublications-
item.grantfulltextnone-
item.languageiso639-1en_US-
item.fulltextno fulltext-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeThesis and Dissertation-
Appears in Collections:生物醫學研究所
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