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研究生(外文):Kai-Dong You
論文名稱(外文):Studies on the Function of Clear Cell Renal Cell Carcinoma- Associated Gene, Heme Oxygenase-1
指導教授(外文):Chih-Hung Hung
外文關鍵詞:Heme Oxygenase-1Renal Cell Carcinoma
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在2006年,腎細胞癌在佔所有的惡性腫瘤中的百分之三,也是最致命的一種腎臟癌類型。成人腎癌中有超過百分之八十五被診斷為腎細胞癌,其中透明細胞型腎細胞癌為最主要的類型,它對於放射性治療、免疫療法和化療均有高度抗性,所以目前以手術切除為主要的治療方式。因此,我們希望藉由研究腎細胞癌相關基因進而了解其形成的分子機制。在先前的研究中,本實驗室的唐賽文先生建構了腎細胞癌以及正常腎臟組織的全長cDNA資料庫。我們採用即時多重聚合酶鏈反應分析法去分析cDNA資料庫,並比對癌細胞與正常組織之間的差異,發現第一型血紅素氧化酶 (heme oxygenase-1,HO-1) 與血管內皮生長因子A (vascular endothelial growth factor A,VEGF-A)在癌細胞中表現量大於正常組織。
HO-1是一種熱休克蛋白,它能因不同的壓力因子而被誘發,例如重金屬、紫外線、氧化壓力及發炎。HO-1是一種速率限制酵素 (rate-limiting enzyme),它能分解血基質 (heme),並產生一氧化碳 (CO)、亞鐵離子 (Fe2+) 和膽紅素(bilirubin)。 在HEK293細胞株中過度表現HO-1可使VEGF-A的RNA和蛋白質表現量上升。在傷口癒合試驗 (wound healing assay) 與細胞穿透試驗 (transwell assay) 的實驗中發現,穩定表現HO-1的細胞株能促進細胞的遷移。在使用HUVEC細胞株的細胞穿透試驗中,發現穩定表現HO-1的細胞株分泌VEGF-A到培養基中,進而促使HUVEC細胞的遷移。
Renal cell carcinoma (RCC) accounts for 3% of all adult malignancies and is the most lethal of the common urologic cancers in 2006. In adults, RCC accounts for over 85% of all diagnosed renal cancers. The clear cell RCC (ccRCC) is major type in renal cancer. It is high resistance to radiotherapy, immunotherapy and chemotherapy. Surgery remains the most effective method of treating renal cancer. Therefore, to investigate whether tumor associated genes are very important for the molecular mechanisms of RCC, full-length cDNA library of ccRCC and normal kidney constructed by Mr. Sai-Wen Tang was investigated. In the cDNA library, Heme oxygenase 1 (HO-1) and vascular endothelial growth factor A (VEGF-A) were found to be up-reglulated in ccRCC tissues compared to their adjacent normal tissues by real-time polymerase chain reaction.
HO-1 is a heat shock protein and can be induced by various stresses, such as heavy metals, ultraviolet radiation, oxidative stress and inflammation. HO-1 is the rate-limiting enzyme in the degradation of heme into carbon monoxide (CO), iron (Fe2+) and biliverdin. Overexpression of HO-1 is upregulated VEGF-A RNA and protein level in HEK293 cell line. Stable expression of HO-1 is promoted cells migration by wound healing and transwell assay. Using HUVEC tranwell assay it was confirmed that the HO-1 stable clone secreted VEGF-A in the medium to promote HUVEC cells migration. In signal transduction pathways, we found that expression of HO-1 increased the phosphorylation of AKT and ERK 1/2. In nucleus fractionation, HO-1 activated the NFκB expression. By cell viability assay, HO-1 can protect cells which were treated with adriamycin. These results showed that HO-1 expressed was upregulated VEGF-A secretion. Taken together, HO-1 could play a role in the progression of RCC carcinogenesis and promote angiogenesis via VEGF-A secretion.
Table of contents:
摘要 1
Abstract 3
Introduction 5
Materials and Methods 9
1. Plasmid Transformation 9
1.1 Preparation of competent cells 9
1.2 Transformation 9
1.3 Plasmid mini-preparation 10
2. Cell culture 10
3. Transient transfection 11
4. Generating HO-1 stable clones 11
5. RNA extraction and reverse transcription 12
5.1 RNA extraction 12
5.2 Treatment of DNA 12
5.3 Reverse transcription of RNA 13
6. Real-time PCR 14
7. Western Blotting 15
7.1 Preparation of Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 15
7.2 Preparation of cell lysates 16
7.3 Electrophoresis 17
7.4 Semi-dry blotting 18
7.5 Immunoblotting 19
8. Cell viability assay 19
9. Confocal Microscopy 20
9.1 Cells preparation and fixation 20
9.2 Immunostaining 20
9.3 Confocal microscopy 20
10. Wound-healing assay 21
11. Cell Migration Assay for HEK293 cells 21
12. Cell Migration Assay for HUVEC cells 21
13. Heme oxygenase-1 (HO-1) induction by hemin 22
14. Heme oxygenase-1 (HO-1) inhibition by Tin-protoporphyrin IX (SnPP) ……………………………………………………………………………..23
15. Subcelluar fractionation 23
Results 24
Overexpression of HO-1 upregulates VEGF-A mRNA expression in HEK293 cells. 24
VEGF-A protein is upregulated by HO-1 expression in HEK293 cells 24
Hemin treatment increased the VEGF-A protein expression 25
Subcellular localization of stable expression HO-1 protein 26
Stable expression of HO-1 enhanced cell migration 26
Stable expression of HO-1 enhanced HUVEC cells migration 27
An inhibitor of HO-1, Tin-protoporphyrin IX (SnPP), decreased the VEGF-A expression 28
Increase of phosphorylation of the AKT and ERK1/2 in HO-1 stable cell line ……………………………………………………………………………..28
Increase of NFκB p50 and NFκB p65 translocation into nucleus in HO-1 stable cell line 29
Protection of cells from adriamycin-induced apoptosis by expression of HO-1 29
Discussion 31
Figures 35
References 61
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