斑馬魚魚鱗表現第一型人類膠原蛋白可增強魚鱗膠原蛋白基質之細胞適應性

Cytocompatibility enhancement for human cell growth on human type I collagen-expressed zebrafish scale

陳漢唐(Han-Tang Chen)

指導教授 ： 吳金洌

共同指導教授 ： 陳志毅

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201801032

摘要

目前全世界中約有超過三千九百萬人因角膜受損導致視力下降或甚至失明，但接受角膜捐贈手術的患者只有不到十五萬人，供需極度不均。因此人工角膜開發勢必成為趨勢。台灣柏登生醫開發人工生物角膜以吳郭魚鱗為基質製成，其成分和角膜相同均為第一型膠原蛋白，並且具有相似的立體三維結構，但卻因移植魚類膠原蛋白而有引發免疫排斥反應以及魚鱗膠原蛋白熱穩定溫度低於人體常溫導致膠原蛋白變性的可能。因此本實驗室提出構想，建立表現人類第一型膠原蛋白之基因轉殖尼羅吳郭魚，得到表現人類第一型膠原蛋白基因之轉基因魚，以人類膠原蛋白低免疫原性及較高的熱穩定性(40℃)的特性，此轉基因魚的鱗片為材料開發新一代之擬人化魚鱗眼角膜可望避免未來臨床試驗中可能面臨之免疫排斥反應以及熱穩定性不足的問題。由於轉基因吳郭魚之建立困難且耗時，因此本研究之宗旨為以第一型人類膠原蛋白基因轉殖之斑馬魚為模式生物探討轉基因魚鱗之細胞適應性是否有顯著提升。本研究以四環素轉錄調控系統建立可持續性表現外源基因之轉基因斑馬魚，並以即時聚合酶鏈鎖反應、免疫組織染色等技術確認人類膠原蛋白之信使核糖核酸與蛋白質之表現後，以人類纖維母細胞、間質幹細胞以及人類角膜上皮細胞三株細胞測試轉基因魚鱗之細胞適應性，並以細胞之貼附、爬行與增生作為判斷適應性好壞之依據，結果顯示，轉基因魚鱗可促進細胞之前期貼附、爬行與生長。在細胞貼附方面，細胞種植後前期細胞貼附的數量與效果可發現細胞在轉基因魚鱗上的數量與貼附面積均比野生種魚鱗大，並且統計後有顯著差異，證明轉基因魚鱗可幫助細胞前期貼附。細胞貼附後兩天內細胞生長數量轉基因魚鱗也高於野生種魚鱗。結論證實，人類第一型膠原蛋白轉基因魚鱗具有較佳的細胞適應性，可作為材料開發生物角膜應用於人體試驗。

關鍵字

魚鱗膠原蛋白基質 ； 人類膠原蛋白 ； 斑馬魚 ； 吳郭魚 ； 細胞適應性

並列摘要

Over 39 million people worldwide suffered from corneal blindness worldwide, but less than 150 thousands corneal grafting have been operated. It’s a pressing issue to find out a substitution replacing the damaged cornea. A biomedical corporation, Body Organ Biomedical Corp. in Taiwan had developed a novel fish scale derived collagen matrix (FSCM) from tilapia as bio-cornea for cornea transplantation, which mainly composed of type I collagen with highly similar three dimensional structure to the human cornea. However, in rat implantation experiment, a mild immune rejection was observed, also the low thermal stability of fish scale collagen might cause partial denaturation under human body temperature. In order to prevent such disadvantage, a “Humanized” scale might be an effective solution. In this proposal, human type I collagen gene which composed of two genes: COL1a1 and COL1a2, were transferred into tilapia and zebrafish embryos, obtaining a transgenic tilapia and zebrafish expressing human type I collagen. Zebrafish was mainly used as a model for following experiments proceed due to the difficulty and time consuming of transgenic tilapia establishment. Human type I collagen expression was confirmed by qRT-PCR and whole mount immunohistochemistry (IHC), respectively. The transgenic fish scale with human type I collagen expression was extracted and proceeded with cell attachment, adhesion, migration and proliferation assay with human cells for cytocompatibility assessment. As results showed, transgenic fish scale could facilitate cell attachment, adhesion, migration and proliferation at early stage. In conclusion, a double transgenic zebrafish stable line with type I collagen expression was established and confirmed. The transgenic scale showed better cytocompatibility comparing to the wild type fish scale, which indicated scale with human collagen expression had opportunity to prevent possible immune rejection and partial denaturation from human clinical trials as a bio-cornea product.

並列關鍵字

FSCMs ； Human type I collagen ； Zebrafish ； Tilapia ； Cytocompatibility

參考文獻

References

1. Brunette, I., et al., Alternatives to eye bank native tissue for corneal stromal replacement. Progress in Retinal and Eye Research, 2017. 59: p. 97-130.

2. Miller, T.D., et al., Validation of cooling effect of insulated containers for the shipment of corneal tissue and recommendations for transport. Cornea, 2013. 32(1): p. 63-69.

3. Jiraskova, N., et al., AlphaCor artificial cornea: clinical outcome. Eye, 2011. 25(9): p. 1138-1146.

4. Shoulders, M.D. and R.T. Raines, Collagen structure and stability. Annual review of biochemistry, 2009. 78: p. 929-958.