Abstract
摘要:子癇前症preeclampsia 的臨床症狀之產生與胎盤產生的抗血管新生(anti-angiogenesis)因子與有關。抗血管新生的結果,造成胎盤發育不足,引起胎兒子宮內生長受限。而這些抗血管新生相關因子的改變,同時可能影響血管擴張能力、影響血管內皮細胞可通透性等造成血管內皮細胞的功能異常,引發母體的水腫及全身性症狀。我們過去的研究已證實Mucin1 在severe preeclampsia 的胎盤有比正常胎盤更高的表現量。由細胞學的研究我們發現Mucin1會增加滋養層母細胞之PTEN表現。此現象的機制與PTEN表現的後續生物效應目前並不清楚。我們比較由Mucin1 overexpression與control trophoblasts所收集的condition media ,發現Mucin1 overexpression會顯著抑制trophoblast原本具有之促進血管內皮細胞 migration 及tube formation等血管新生能力。我們也發現Mucin1會影響condition media中血管新生蛋白質的表現模式。由於PTEN的表現是已知重要的血管新生抑制訊息。因此在本計畫中,我們將探討Mucin1如何增加滋養層母細胞之PTEN表現之分子機制,並進一步探討PTEN表現是否具有抑制血管新生因子產生及滋養層母細胞的增生等生物效應。本次研究方法與策略包括:第一年:以臨床胎盤組織檢體探討Mucin1與PTEN表現的臨床意義並釐清Mucin1調節PTEN的分子機制。研究設計與施行的策略為:1. 進行Mucin1、PTEN、血管密度、細胞增生的免疫組織染色與量化分析,比較正常與子癇前症組別中的表現差異、此部分本人已經有充足的組織檢體只要補充IRB的說明。2. 以細胞模式運用Rapamycin及Cyclosporin A探討Mucin1對於PTEN表現是轉錄層次或轉譯層次之調控。3. 探討Mucin1是否經由microRNA調控PTEN表現,經以目前已經知道的miRNA PTEN (mir19a, mir21, mir25, mir28)為標的。偵測它們在Mucin1處理之細胞的表現並以anti-sense ODN驗正調控PTEN功能。4. 構築PTEN promoter based reporter gene,搭配decoy ODNs探討Mucin1
Abstract: The clinical syndrome of preeclampsia is related with the anti-angiogenesis factors from placenta. Anti-angiogenesis effect may influence on placenta development and result in intrauterine growth restriction. Besides, these anti-angiogenesis factors may interfere to the vessel dilatation, change the endothelial permeability and cause the vascular dysfunction, then resulting in generalized edema. Our past study has revealed that Mucin1 is overexpressed in the placenta of severe preeclampsia. To clarify the role of Mucin1 in the angiogenesis capability of trophoblasts, we have established a Mucin1 over-expression trophoblast cell line. The conditional medium from the Mucin1 over-expression trophoblast cell line expressed the lower angiogenic capability rather than the wild type trophoblast cell line, by using an angiogenesis protein antibody array. The results revealed that Mucin1 overexpression significantly changed the protein expression pattern of angiogenesis protein. We also found that mucin1 could enhance the PTEN expression. PTEN has been found to play critical role in anti-angiogenesis. In this project, we will investigate the role of Mucin1 in regulating of PTEN expression and suppressed angiogenetic factor expression in the trophoblasts.The study design and major methods are:First year: 1. Investigate the clinical correlation among the MUC1, PTEN and angiogenesis (micro vessel density) in placenta tissue from normal and preeclampsia2. Investigate the transcriptional or translational regulation of PTEN by Mucin1 with rapamycin and cyclosporin A, respectively. 3. Construct the PTEN promoter based reporter plasmid, combined with the decoy ODN strategy to evaluate the transcriptional factors (possible NF-kB and AP-1) involved in the MUC1 mediated PTEN gene regulation. 4. Screen out the possible mirRNA (miRNA PTEN (mir19a, mir21, mir25, mir28)) which is involved in the MUC1 mediated PTEN expression by qRT-PCR. 5. Evaluate the signaling transduction pathway (such as PI3K/AKT by inhibitor LY294002, MAPK/p38 by SB203580 and MAPK/ERK by PD98059) that is involved in the regulation of PTEN by Mucin-1.Second year:1. Angiogenesis effect of PTEN dependent angiogenesis factor will be verified by angiogenesis assays.2. Verify the expression of MUC1,PTEN and angiogenesis in C57BL/6 mice model in the trophoblast from different stage of pregnancy.3. Manipulate MUC1 level in pregnant mice by recombinant mouse MUC1 injection .The angiogenesis of placenta will be determined by micro vessel density determination.5. Evaluate the role of specific miRNA PTEN in the MUC1-PTEN regulation. The expression of PTEN will be determined by IHC. The angiogenesis of placenta will be determined by micro vessel density determination. This study will provide important information for the cellular mechanism of Mucin1 modulated PTEN, realize the possibility of miRNA PTEN molecular targeting and the role of PTEN in the angiogenesis promoting capability of trophoblasts.
Keyword(s)
黏液蛋白
滋養層母細胞
PTEN
子癇前症
mucin
trophoblast
PTEN
preeclampsia