以短暫基因表現策略將小鼠體細胞再程序化成生殖細胞之研究

Abstract

根據世界衛生組織研究統計，全球不孕症發生率約佔15%，且大多需人工輔助生殖，惟無精症或配子無法成熟患者迄今仍無生殖輔助策略可施，若能釐清體細胞於體外再程序化為生殖細胞之相關機轉，即可藉由體細胞誘導為生殖細胞予以輔助。本研究擬以NIH3T3細胞株（染色體XY）探討終端分化之體細胞能否依序透過短暫基因表現（transient gene expression）生殖細胞發育時期之不同關鍵基因，使細胞進行再程序化（reprogramming）回到相對原始狀態，並轉分化（trans-differentiation）為生殖細胞，同時釐清其相關分子機轉。 本研究擬將生殖細胞不同時期表現之基因分別構築於三個不同質體中，轉染時間係依照小鼠體內生殖細胞相關之基因變動進行調控，試驗擬依序將小鼠生殖細胞前期、中期及後期表現之基因以漸進式誘導方式將質體短暫轉染（transient transfection）至細胞內。其中第一組含綠螢光報導基因及始基生殖細胞（primordial germ cell, PGC）前期表現基因-T factor（Brachyury）、Blimp1、Prdm14及Lin28之質體，是希望讓NIH3T3細胞模擬回到胚胎發育上胚層 (epiblast) 剛形成PGC之狀態;第二組含紅螢光報導基因係三個生殖細胞相對中期之重要基因-Stella、Vasa及Dazl，期細胞能依序發育為遷移時期之生殖細胞，最後一組則加入含藍螢光及兩個減數分裂相關之基因- Stra8及Sycp3，期能促使細胞進一步進行減數分裂。轉染後分別收集不同天數之各組細胞，進行物理化學特性分析。試驗結果顯示轉染成功之細胞有如同PGC於體外培養時之明顯漂浮特性，細胞型態完整並持續表現螢光，初步推測誘導之細胞可能往PGC之譜系發展。轉染成功細胞之多能性基因-Oct4、Sox2和Nanog及受體基因-BmpR1A、BmpR1B及FGFR表現量皆有較未轉染者高之趨勢，而三胚層之不同代表基因也維持在很低的表現量，此外，生殖細胞相關基因-Tfap2c、Dnd1、Rhox9等之表現量與未轉染之細胞相比較亦有上調趨勢，減數分裂基因-Dmc1及Spo11之表現量有上升的趨勢。依上述結果顯示短暫基因轉染不同生殖細胞時期基因誘導之體細胞株，除了可能經歷再程序化外，同時也轉發育為生殖細胞譜系細胞，並啟動減數分裂相關之機轉。此外，我們進行了免疫染色觀測細胞蛋白質層次上之變動，結果發現轉染任何一組別之質體，經過七天的培養後皆能以免疫染色觀測到OCT4、TFAP2C及DMC1蛋白的表現，未來將進一步分析染色體套數之改變，將可更完整釐清誘導之細胞染色體套數是否有變動。 總括上述，研究結果顯示經過短暫基因轉染技術轉染生殖細胞不同時期表現基因之細胞，除了可誘導體細胞經歷再程序化回到相對原始之狀態外，並顯示往生殖細胞譜系發展之跡象。此小鼠體細胞誘導為生殖細胞之平台建立有助於了解體細胞往生殖細胞發展之相關機轉，未來可提供人類體細胞誘導生殖細胞之參考，期望將來可藉由體細胞於體外誘導為生殖細胞之技術輔助無精症患者治療。

According to the World Health Organization statistics in research, infertility affects an estimated 15% of all couples globally and requires assisted reproductive technology. In current technology, we still have no effective resolution to implement, on patients with azoospermia or one’s gametes cannot reach the maturity stage. If we can clarify the relevant mechanisms of reprogramming somatic cells into germ cells lineage in vitro, we can assist them by reprogramming somatic derived cells into germ cells. We are fully committed to explore the feasibility of terminally differentiated somatic derived cells NIH3T3 (chromosome XY) trans-differentiation into germ cell lineage. We hope to clarify whether we can use transient transfection technology to the sequential induction of different sets of germline critical genes, according to the developmental stages. We want to induce cells into a relatively primitive stage, directly transdifferentiate into germ cell lineage cells and clarify their related molecular mechanisms. we intend to construct the plasmid that contains different sets of genes, according to the developing stage of germ cells. The optimal transfection strategies are similar in vivo gene expression profiles in mice. In this study, we used a novel strategy for somatic derived cell by using the sequential induction in the early-term, middle-term, and late-term germ cell critical gene on the development. First of all, we overexpress plasmid in the NIH3T3 by early-term primordial germ cell gene, which contains green fluorescent reporter genes, and the early period primordial germ cells (PGC) genes - T factor, Blimp1, Prdm14, and Lin28. We hope to make NIH3T3 simulate the stage of epiblast forming PGC in the embryonic development. The second group contains a red fluorescent reporter gene and three important genes of the middle-term germ cell lineage gene - Stella, Vasa, and Dazl. We expected that the cells can develop into germ cells in the migration period through the sequential induction. Next, we regulate the plasmid contain blue fluorescence and two meiosis-related genes -Stra8 and Sycp3, which can promote further meiosis. After transfection, we collected cells of different days and different treatment groups to analyze the physical and chemical properties in different aspects. The results showed that the transfected cells had the same suspension characteristics as the germ cell culture in vitro. We observed that the cell membrane is complete and continues to express fluorescent genes. Therefore, we preliminarily speculate that the induced cells may develop into the germ cell lineage. According to our results, the transfected cells' pluripotency genes - Oct4, Sox2, Nanog, and the receptor genes - BmpR1A, BmpR1B and FGFR are higher than those without transfection. The genes of the three germ layers are also maintained at a very low expression level. In addition, the expression levels of germ cell-related genes ( Tfap2c, Dnd1, Rhox9, etc.) also have a significant upward trend compared with control group. Furthermore, the expression of meiotic genes - Dmc1 and Spo11 has an upward trend. These results implied the somatic derived cell transfected with different sets of plasmids had undergone reprogramming. Then, we performed immunostaining to detect the cell properties at the cellular protein level. We found that no matter which group of plasmids we transfected, the expression of OCT4, TFAP2C and DMC1 protein can be observed in every group by immunostaining after seven days of treatment. In the future, we will analyze the number of chromosome sets. In days to come, we will clarify whether the sets of the chromosome have changed. In summary, our study shows that using transient transfection to somatic derived cell, the cell underwent reprogramming, developed into germ cell lineages, and initiated meiosis mechanisms. We hope to clarify the mechanism of somatic cell inducing into germ cells in the future to help treating azoospermia disease.