2011-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/656215摘要：人類的基因體計劃已於2003 年完成解密，但科學家發現基因序列並無法解釋許多細胞生理的正常運作，後基因體時代於是到來。除了轉譯醫學外，表現遺傳學(Epigenetics)的基因調控作用占了非常重要的角色。特別的是，許多固定序列的真核細胞，在特定的環境下會有不同的表現，表現遺傳學在於解釋基因與環境的互動上更加有其重要角色。表現遺傳學調控了幾乎所有的生理過程，包含早期細胞分化、胚胎發育、器官生長，以及病態的過程(如腫瘤的發生)。主要調控機制為利用DNA 甲基轉化脢，將基因的啟動子區域中的 CpG island 位置甲基化，藉此阻礙轉錄因子與 DNA 結合，進而導致基因沈默(silencing)。人類的癌症發生與滋養層細胞的侵襲有許多共同的特點，包括異常的血管增生、或基質金屬蛋白脢路徑的活化均有高度類似之處。在最近幾年內，科學家又猜測癌症與滋養層細胞的侵襲均牽涉到許多基因的異常甲基化(主要是抑癌基因)，導致有關侵襲路徑的基因不受牽制而產生高度侵襲。可惜這在滋養層細胞方面的研究非常有限；而在先進國家中，母體的子癲前症是一個非常重要引起母體死亡率及罹病率的原因，雖然致病原因並不明瞭，但滋養層細胞對母體蛻膜的侵襲能力不足，被認為是一個重要的導因。基於以上的理由，本計劃著重於甲基化對滋養層細胞的侵襲相關基因之探討。第一年計劃將針對初期懷孕引產所得的絨毛，各自純化為絨毛部及間質部的品系，再與購得的3A‐sub‐E，JAR,JEG‐3 去進行全基因體的DNA 甲基化晶片分析，期望可以發現侵襲或惡性轉變過程中牽涉的基因甲基化轉變的熱點，再進一步分析何種基因在此過程佔有重要角色。第二年的計劃中，我們援用Caniggia 等在2000 年建立的滋養層細胞的侵襲模式，將細胞培養加入TGF3 以減少細胞侵襲能力(模擬子癲前症的狀況)、或加入TGF3的抗體以增加細胞侵襲能力，利用這樣的培養模式，去檢視是否加入甲基化抑制劑(AZA)，可以導致侵襲能力的改變，去反向驗證滋養層細胞的侵襲與DNA 甲基化的相關，同時藉由短訊息干擾核苷酸去大幅減低DNA 脫氧核糖核酸脢各基因家族活性，看看是那一個DNA 脫氧核糖核酸脢在侵襲路徑扮演重要角色。此計劃第三年將比較正常與子癲前症的孕婦生產所得的胎盤，比對其全基因體的甲基化差異。同時將子癲前症胎盤的絨毛以Matrigel 做體外培養，並加入甲基化抑制劑，看是否可以改變子癲前症胎盤的絨毛侵襲能力，並驗證其是透過何種基因的調控。本計劃除可以深入探討滋養層細胞的侵襲機轉，更可能對子癲前症發生的原因有更進一步的認識，期望可以為子癲前症的治療提出新的研究方向。<br> Abstract: The working draft of the human genome project has been completed since 2003.However, the scientists found that the DNA sequence cannot solely explain many of thephysiological process of eukaryotic cells, therefore foretelling the coming of post-genomicera. In addition to translational medicine, the gene functions regulated by epigeneticsbecome an essential part to titrate the cellular function. Remarkably, many cells with distinctgenetic sequence may exert different function, indicating the interplay of gene andenvironment. Among them, epigenetics may play an essential role to regulate the interactionbetween genes and their environment. The process includes the modifications of CpG islandof the promoters of gene, usually through the methylation, to silence the expression ofcertain genes.There are many similarities between cancer cell invasion and trophoblast invasion,including abundant angiogenesis and the activation of MMP pathway. Recently, theresearcher also postulated that the invasive growth of tumor and blastocyst implantation mayshare the similar mechanisms of epigenetic regulation. However, there are very few studiesconcerning the invasion pathway of trophoblast. Notably, preeclampsia is a major cause thatcontributes to the maternal mortality and morbidity in the developing countries. Though it’snot fully understood, shallow invasion of trophoblast is postulated as an importantphenomenon of preeclampsia.Based on the above reasons, we investigate the relationship of epigenetics andtrophoblast invasion in this 3-year project. In the first year, we purify the villous trophoblastand intermediate trophoblast from the villous tissue of first-trimester abortion. We alsopursed 3A-sub-E and JAR, JEG-3 cell lines for compared. Whole genome methylation DNApatterns were compared between these immotile, invasive, pre-malignant and malignanttransformed trophoblast. In the second year, we first used the established model fromCaniggia (2000) which use TGF3 to inhibit the trophoblast invasion (mimicking the in vivocondition of preeclampsia) and TGF3 antibody to rescue this condition. These culturesystem are further treated with 5'-aza-2'-deoxycytidine (AZA), a potent methylation inhibitor,to see if there any phenotypic /invasive behavior changes. If occurred, siRNA of individualisoforms of DNMs (DNA methyltransferase) will be used to decrease their gene functions.Then we are able to see which DNMT is responsible for these phenotypic changes. In theproject of the last year, we compare the genome-wide DNA methylation patterns between thesample of placenta tissues from preeclampsia and normal control. A villous explant model isalso used to mimic the preeclampsia in vivo. We will interrogate if the adding AZA willchange the behavior of preeclamptic trophoblast, and to see which transcripts areupregulated to accomplish this change. Taken together, this 3-year project will investigatethe important steps of trophoblast invasion, and may also shed a light to direct thedevelopment of new treatment for preeclampsia.