| 摘要: | 第一部分:托吡酯在斑馬魚中母系遺傳的途徑
斑馬魚(Danio rerio) 為近幾年發育生物學興盛的動物模式之一，以斑馬魚做為實驗動物已經知道有許多益處，例如胚胎量產大、快速發育和與人類的遺傳相似性高，故被廣泛運用於動物遺傳、胚胎發育、藥物篩選、毒藥物試驗及人類相關疾病研究。
托吡酯 (topiramate) 是一種通常用於治療兒童和成人的癲癇症狀以及預防偏頭痛的口服藥物。食用托吡酯常見的副作用包括皮膚感覺異常症狀、體重減輕、食慾不振、疲憊疲勞、腹痛、脫髮、注意力不集中等。懷孕女性在服用托吡酯後可能會導致胎兒/新生兒造成口腔裂縫出生缺陷(唇裂/顎裂)的風險增加。
在本實驗過去與淡江大學陳曜鴻老師共同研究發現，餵食托吡酯後的雌性斑馬魚的卵巢功能受到影響，進而發現其後代有顏面部骨骼發育異常的現象，因此在本次研究中，我們想要探討是否雌性斑馬魚的卵巢在基因傳遞 (genetic transmission) 的功能上受到影響甚至阻礙，而造成子代骨骼發育的缺陷。我們首先連續以托吡酯餵食雌斑馬魚七天後於第八天並收集其卵巢、未受精卵子、以及所產下的胚胎，偵測各骨骼發育相關基因表現量。我們發現服用過托吡酯後母魚各項基因傳遞至卵子及孵化後的胚胎皆受到影響，其中bmp2a、bmp2b、runx2b的基因傳遞量皆呈現下降趨勢。未來將進行免疫組織染色以確認上述目標基因於卵巢組織中蛋白質表現，進而推論因服用托吡酯而影響母代基因傳遞至子代的路徑。一旦模式建立，不但使我們瞭解斑馬魚中母代基因傳遞的方式，並且能確定托吡酯導致基因傳遞的功能受損的作用機制。
第二部分:天然藥物對斑馬魚胚胎的影響
天然藥物的使用皆有其藥物的藥理作用與作用途徑。藥物使用於人體前需要經過生物實驗以確保藥物的有效性及對生物的傷害性，因此使用動物檢測是藥物上市前不可或缺的一部分。斑馬魚近年來在全世界廣泛的用於研究脊椎動物的胚胎發育、疾病研究、藥物篩選、及毒物測試。
研究取用斑馬魚胚胎，並給予氯化鋰（LiCl）、天然藥物（銀杏），研究斑馬魚胚胎在給予藥物後會產生之發育狀態與型態變化，及對於使用藥物劑量又對胚胎造成的影響，進行藥物對於斑馬魚胚胎毒性的效果檢測。實驗主要使用氯化鋰試劑作為引發Wnt路徑，再分別使用天然藥物來觀察對斑馬魚胚胎影響性。在實驗前一天將公魚與母魚分開放置，並於實驗當天早上再將斑馬魚配對並取得受精卵後，將實驗藥物加入斑馬魚胚胎中48至72小時，並在每24小時觀察斑馬魚卵的成長情況以及死亡情形。觀察胚胎適合的濃度範圍。之後再對胚胎進行蛋白質的抽取、蛋白質的定量分析、正十二烷硫酸鈉－聚丙烯醯胺膠板電泳鑑定蛋白質、西方墨點法實驗方法，來分析檢測特定蛋白質的表達情況。
結果發現，氯化鋰會使Wnt路徑中β-catenin蛋白質濃度升高，在濃度高於200 mM會造成胚胎異常、高死亡率。再加入銀杏後能有效抑制β-catenin蛋白質濃度，代表著銀杏可能存在著降低β-catenin蛋白質的趨勢。
Part1. Genetic pathway of maternal transmission in Topiramate-treated zebrafish model
Zebrafish (Danio rerio) is one of model organism for developmental biology in recent years. As an experimental animal, zebrafish has many advantages. For example, its embryos are easy to collect and reproduce. Zebrafish grows rapidly and has similar heredity to humam beings. Therefore, it is widely used in animal genetics, embryonic development, drug screening, toxic drug testing and human-related diseases research. To understand the potential effects of topiramate on female zebrafish, we subjected adult female zebrafish topiramate and focused on the genomic changes of adult females and their offspring.
Topiramate is an oral drug used to treat epileptic symptoms in children and adults and to prevent migraine. Common side effects of using topiramate include abnormal skin sensation, weight loss, loss of appetite, fatigue, abdominal pain, hair loss and inattention. Pregnant women taking topiramate may increase the risk of cleft lip or cleft palate in fetuses, we have found that the ovarian function of female zebrafish is affected after fed on topiramate and abnormal facial skeletal development in offsprings. In this study, we aimed to explore whether ovaries are affected or even hindered in the function of genetic transmission. First, we continuously feed female zebrafish on topiramate for a week. Then, we collected ovaries, unfertilized eggs and hatched embryos to detect the expression of skeletal development-related genes. We also compared the expression of skeletal development-related genes and the percentage of genes transferred to unfertilized eggs or embryos. We found that after feeding topiramate, several genes transferred to eggs and hatched embryos from female fish were decreased, such as bmp2a, bmp2b, runx2b. We will stain these tissue to detect the protein expression of the target genes in ovarian tissues and to establish a zebrafish model that demonstrates the transmission of maternal genes after topiramate administration. Once the model is established, we can not only understand the mode of maternal gene transfer in zebrafish, but also determine the mechanism of topiramate resulting in impairment of genetic transmission.
Part 2. Effects of herbal medicine on zebrafish embryos
The use of natural drugs has its pharmacological effects and pathways. We need to carry out biological experiments before drugs are used in humans to ensure their effectiveness and bioharmfulness. Animal testing is also an indispensable part for the experiments. In this research, we use zebrafish which is widely used in embryonic development of vertebrates, disease research, drug screening and toxic drug testing in recent years. The zebrafish embryos were injected with lithium chloride (LiCl) and natural medicines (Ginkgo biloba) to discuss their developmental status, morphological changes, the influences of drug dosage on embryo death and detect drug toxicity on zebrafish embryos.
Lithium chloride was used as the initiation pathway of Wnt and natural medicines were used to observe the effects on zebrafish embryos. In order to let the zebrafish lay eggs, we separated male and female fish on the day before the experiment and mixed them the next day in the morning. After that, we immersed the experimental drugs in zebrafish embryos for 48 to 72 hours and observed the growth and death of zebrafish eggs every 24 hours. We tested the toxicity of zebrafish embryos first to find the suitable concentration range of zebrafish embryos. Then, we exrtactsed protein from embryos to analyze quantitatively, identify by SDS-PAGE gel and perform a western blot so that we can know the expression of specific proteins.
The results showed that lithium chloride could increase the concentration of β-catenin protein in Wnt pathway. When the concentration was higher than 200 mM, it could cause abnormal embryos and high mortality. Adding Ginkgo Biloba could effectively inhibit the concentration of β-catenin protein. This indicates that Ginkgo biloba might reduce β-catenin protein. |
