肝臟X受體激活物T0901317抑制人類臍靜脈內皮細胞血管新生之研究

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研究生：	賴至人 Lai, Chih Jen
論文名稱：	肝臟X受體激活物T0901317抑制人類臍靜脈內皮細胞血管新生之研究 The Study of Liver X Receptor Agonist T0901317 Suppresses Angiogenesis of HUVEC Cells
指導教授：	褚志斌 Chuu, Chih Pin 汪宏達 Wang, Horng Dar
口試委員:	陳雅雯 Chen, Ya Wen 張中和 Chang, Chung Ho 褚志斌 Chuu, Chih Pin 汪宏達 Wang, Horng Dar
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	42
中文關鍵詞：	血管新生
外文關鍵詞：	Angiogenesis
相關次數：	點閱：2 下載：0
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LXR對於膽固醇、脂質的合成以及醣類的代謝等體內平衡作用都扮演著相當重要的調控角色，因此LXR在心血管疾病的治療上是個十分有潛力的標的。LXR是一種核受體，有兩種isoform，分別是LXR 和 LXR ，可由激活物活化，與RXR形成dimer之後結合在DNA上藉此調控下游的基因表現。我們由之前的實驗結果以及文獻得知LXR的激活物可以有效抑制攝護腺腫細胞的生長乃至於減緩攝護腺癌細胞的轉移進程。我們實驗發現在HUVEC細胞中加入LXR的激活物T0901317能有效抑制血管新生的生長。在HUVEC 細胞migration實驗和雞胚胎囊膜實驗中也看到加了LXR激活物T0901317能夠明顯降低HUVEC細胞的移動能力以及雞蛋胚胎血管的生長。我們從LXR的標的基因篩選出幾個有可能的基因，發現T0901317可能是藉由Apolipoprotein D這條路徑來調控血管新生的情況。透過angiogenesis protein array，我們也發現到LXR的激活物T0901317對於一些血管新生的蛋白以及發炎反應的蛋白都有相當程度的調控作用，而血管新生與發炎反應更與癌細胞發展和腫瘤生長息息相關。我們正嘗試著去找出更全面、更完整的訊號調控路徑，以建立更完整的LXR調控血管新生的網絡及資訊。

Liver X receptors (LXRs) are important sensors and regulators for cholesterol, fatty acid, and glucose and play essential roles in the development and progression of cardiovascular diseases. Liver X receptors (LXRs) belong to the nuclear superfamily and contain two isoforms: LXR and LXR. LXR dimerizes with RXR in response to the stimulation of either one's agonists. We previously reported that treatment with LXR agonists suppresses the tumor growth and the disease progression of prostate cancer. In this study, we examined if treatment with T0901317, the most potent LXR agonist, may suppress angiogenesis. The results of tube formation assay indicated that treatment with T0901317 inhibited the tube formation of HUVEC cells. T0901317 treatment suppressed migration of HUVEC cells as determined by migration assay and inhibited angiogenesis in chorioallantoic membranes assay in vivo. Angiogenesis protein array revealed that T0901317 treatment suppresses inflammation response. Our results suggested that activation of LXR may interfere angiogenesis via induction of inflammation-related proteins and LXR target genes as well as decrease of angiogenesis regulatory proteins. We discovered that Apolipoprotein D (ApoD) may be a potential target for tumor angiogenesis inhibition. Based on our findings, we believe that administration of LXR agonist T0901317 may be a potential treatment for cancer agiogenesis.

誌謝 ……………………………………………………………….............................I
Abstract……………………………………………………………………………..II
中文摘要…………………………………………………………………………...III
Chapter 1 Introduction………………………………………………………….1
1.1    Liver X receptors (LXRs)………………………………………………………….1
1.2    Liver X receptors agonist………………………………………………………….3
1.3    Camcerous versus physiological angiogenesis…………………………………….3
1.4    The formation of tumor blood vessels……………………………………………..4
1.5    Angiogenesis and tumor progression……………………………………………...5
1.6    Human Umbilical Vein Endothelial Cells…………………………………………7
1.7    Flow chart………………………………………………………………………….8
Chapter 2 Materials and Methods……………………………………………9
2.1 Cell culture………………………………………………………………………...9
2.2 Tube formation assay……………………………………………………………...9
2.3 Migration assay…………………………………………………………………..10
2.4 Chorioallantoic Membranes (CAM) Assay………………………………………10
2.5 Angiogenesis protein assay………………………………………………………11
2.6 Western blot analysis……………………………………………………………..12
2.7 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)……………….12
2.8 Statistical Analysis……………………………………………………………….13
Chapter 3 Results..................................................................................................14
Chapter 4 Discussion…………………………………………............................16
Chapter 5 Future Direction…………………………………………...............20
Chapter 6 Figures……………………………………………………..................21
Figure 1.A Treatment with LXR agonist T0901317 suppresses HUVEC angiogenesis in vitro (tube formation assay)……………………………………………………….21
Figure 1.B Treatment with LXR agonist T0901317 suppresses HUVEC angiogenesis in vitro (tube formation assay)……………………………………………………….22
Figure 2.A T0901317 suppresses angiogenesis in vivo (Chorioallantoic Membranes Assay, CAM)…………………………………………………………………………23
Figure 2.B T0901317 dose-dependently suppressed the migration of HUVEC cells….………………………………………………………………………………..24
Figure 3.A&B Select LXR target gene APOD as candidate by qPCR and WB…......25
Figure 3.C&D Select LXR target gene APOD as candidate by qPCR and WB……..26
Figure 4.A Overexpression of ApoD suppresses the tube formation of HUVEC cell.27
Figure 4.B Overexpression of ApoD suppresses the tube formation of HUVEC cells28
Figure 4.C Protein level of HUVEC overexpressing ApoD………………………….29
Figure 4.D ApoD is a potential tumor suppressor in different types of cancers……..30
Figure 5.A HUVEC cell lysates for angiogenesis array assay……………………….31
Figure 5.B HUVEC cell lysates for angiogenesis array assay……………………….32
Figure 5.C HUVEC cell lysates for angiogenesis array assay……………………….33
Figure 6.A.1 Treatment with T0901317 increased Thrombospondin-1 and Angiostatin in HUVEC cells……………….……………………………………………………...34
Figure 6.A.2-3 Treatment with T0901317 increased Thrombospondin-1 and Angiostatin in HUVEC cells.………………………………………………………...35
Figure 6.B.1 Treatment with T0901317 suppressed MMP-8, MMP-9, VEGFA, and VEGFC in HUVEC cells…………………………………………………………….36
Figure 6.B.2-5 Treatment with T0901317 suppressed MMP-8, MMP-9, VEGFA, and VEGFC in HUVEC cells…………………………………………………………….37
Chapter 7 References…………………………………………………………...38

                                

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