| 摘要: | 斑馬魚(Danio rerio)為近幾年發育生物學興盛的動物模式之一，相較於其他脊椎動物，其特點為生長週期短、斑馬魚胚胎透明容易觀察、飼育容易、體外受精、且突變種類多，故廣泛被運用在動物遺傳、胚胎發育、藥物篩選、毒物測試以及人類相關疾病的研究。例如，在研究肺結核的文獻中，作者使用斑馬魚的胚胎為模型且使用奈米粒子投遞藥物，並進行觀察與研究在斑馬魚胚胎上治療肺結核細胞(1)。
天然藥物的運用都有其藥物作用途徑與反應途徑，但並非都對生物體沒有任何的傷害與損壞。任何一種藥物在使用在人體之前，都會反覆進行實驗與動物試驗，進而觀察其藥物是否有副作用與傷害性。在本實驗中，我們所使用的38號藥劑(hm38)為天然植物萃取物，利用人體癌症細胞經過生長以及細胞凋亡的實驗中得知hm38具有抗癌的效果，但並不知道對於hm38的作用機制是否對細胞生長發育無其他的傷害或影響。所以為了確認hm38抗癌的效果以及對於生物體的毒性，我們分別加入不同濃度的hm38 在斑馬魚胚胎的培養液中，測試藥劑對於斑馬魚胚胎是否能引發抗癌機制或有其他的傷害。
我們在實驗前一天將公魚母魚分開放置一晚，在實驗當天早上將前一晚分開放置的公魚母魚混合放置，促使斑馬魚產卵，並將 hour post fertilize (0 hpf)的胚胎分別加入不同濃度的hm38: 0%、0.5％、2.5%、10%中培養72小時，並且每24小時進行觀察斑馬魚胚胎有無形態上的變化。經初步觀察，當hm38濃度增高，斑馬魚胚胎死亡率也大幅提升，雖然在2.5%及10%皆有延遲發育的現象，但在0.5%濃度下胚胎沒有顯著的發育遲緩。其中有趣的是，若在72小時後將所有的濃度移除hm38，胚胎又會繼續發育。其他非特異性的變異，如心包膜水腫則偶爾會發生，顯示hm38對於斑馬魚早期的發育並沒有顯著的毒性。除此之外，在加入hm38後72小時我們抽取斑馬魚胚胎的蛋白質與RNA進行實驗，hm38濃度越高時，會使 protein p53增生越快，而會抑制 protein BCL2的反應，進而使細胞生長速率變慢，細胞凋亡速度加快。
Zebrafish (Danio rerio) is one of the animal models for developmental biology that flourished in recent years. Compared to other vertebrates﹐zebrafish is characterized by short growth cycle, transparent embryos, easy breeding, and many kinds of mutation for genetic screening. It is widely used in embryonic development, drug screening, toxicology testing and identifying human-related diseases.
Every natural medicine has its pharmaceutical pathways; however, it may be harmful when treated in any condition. Before launching drugs for any purposes, repeated experiments and animal trials are necessary to examine if drugs have any side effects and harmfulness. In previous experiment, heral medicine 38, crude extract of one of native herbs in Taiwan, has been identified some anti-cancer effects on human lung cancer cells. It inhibited cell growth and increased cell apoptosis, Which indicates hm38 has potential anti-cancer activity.
We separated male and female fishes at night before setting up ezperiment. In the next morning, fishes were prompting and hour-post-fertilization (0 hpf) embryos were collected. Different concentrations of hm38: 0%, 0.5%, 2.5%, 10% were treated on embryos for 72 hours. In our preliminary, we observed that when increasing hm38 concentration, embryos have phenomenon of delayed development at 2.5% and 10%, and mortality increased dramatically; however, when treated embryos with 0.5% hm38, thereis no significant toxicity and growth retardation. Other non-specific variations may occur during treatment, such as the pericardium edema, which indicates hm38 for early zebrafish development has no significant toxicity. We subsequently test the signal molecules involving in anti-cancer pathway. We perform realtime-PCR analysis to screen the potential pathway that is affected by hm38. The mRNA expression levels of p53 is induced while BCL2 is significantly reduced, which suggests hm38 induces apoptosis through p53-mediated pathway by activating p53 and inhibiting BCL2 function. On other hand, we test the gene expression of SOX4a and 4b. While SOX4 is an oncogene, there is mounting evidence suggesting a role for SOX4 involvement in tumorigenes. Based on the mRNA expression pattern, SOX4a and 4b may exert potential anti-cancer progression function. In addition, protein level of caspase-3 and p53 are increased after 72 hrs treating 0.5% hm38, which correspond to our realtime-PCR results.
According to our results, this study demonstrates the feasibility of this novel combinational approach and shows that hm38 has potential to be anti-cancer agents. |
