摘要:在生物醫學領域中,利用實驗動物模擬人類具有類似的病理現象,進行生物醫學、行為試驗,而其產生的先天或誘發性病理變化,能用以作為疾病發生原因與治療療效參考,此稱作人類疾病之動物模式。適合的動物模式是進行基礎醫學的研究成功的關鍵。自生物醫學發展至今,雖然囓齒類動物扮演著研究模式的最多數,然而囓齒動物在生理、解剖、遺傳與壽命等各方面與人類有明顯差異性,因此模擬某些特定疾病以囓齒類動物為模式並不是最合適。而在各類不同的實驗動物中,豬(Sus Scrofa)為連結囓齒動物模式與人類疾病研究或實際治療應用的最佳試驗與研究動物模式,在疾病研究的演進上扮演了一個重要且關鍵的角色。
蘭嶼豬屬於臺灣原生小型豬。1975至1980年畜產試驗所因應「發展豬隻供作醫學研究之用」的政策,開始進行開發實驗動物豬的育種及保種計畫。臺灣大學畜牧學系(現今的動物科學技術學系)將蘭嶼豬(母)與Landrace豬(公)雜交選育出具白色毛皮的李宋豬(75%蘭嶼豬,25% Landrace,亦屬迷你豬)。2008年,在選育族群中出現無眼球(Anophthalmia)與缺虹膜(Aniridia)兩種表現型的缺失李宋豬,由配種操作研究確定為遺傳缺失。
小鼠與大鼠皆有類似無虹膜、無眼球的遺傳缺陷或基因剔除鼠模式產生。但由許多研究亦得知,囓齒動物的解剖構造、生理、壽命與分子遺傳皆與人類有相當差距。有些眼球發育缺失,囓齒動物研究結果無法接近於人類。由人為基因突變研究發現,目前調控囓齒類動物無虹膜、無眼球發育相關基因,主要與Pax6、Sox2、Otx-2、Six3、Rax與Lhx2等等基因有關。因為,這些外表型與李宋豬無虹膜、無眼球之缺陷相似且由我們配種研究結果顯示,李宋豬的眼球發育缺失乃由幾個基因調控之簡單遺傳性狀且可能有基因表現劑量差異的影響。於是,我們提出假設:我們的李宋豬無眼球的缺失可能是由於Pax6、Sox2、Otx-2、Six3、Rax與Lhx2等基因發生缺失而引起,而無虹膜可能由上述基因表現劑量差異發生缺陷所引起。
Notara等人(2011)由組織學與外科手術證實豬相當適合做眼球受傷、組織移植與眼球上皮細胞發育(如幹細胞)研究。因此,藉由這些眼球發育缺失迷你豬的解剖生理學與分子遺傳學的研究,可建立一個適合研究眼睛發育與疾病治療的中大型實驗動物模式。去年我們完成Pax6、Sox2與Otx-2基因序列檢測,2014年度我們計畫(一)期望利用育種計劃,建立可提供李宋系迷你豬品系穩定產生無虹膜、無眼球之缺失純系品種;(二)建立無虹膜、無眼球及正常眼球發育之純系品種之遺傳學基礎資料。檢測Six3、Rax與Lhx2等基因序列缺失與計算與眼球缺失發生之相關性;(三)以組織免疫螢光染色技術(組織切片)確定無虹膜、無眼球及正常李宋豬之眼球上皮細胞、組織的結構、細胞分布與人類的差異。目前國際上亦無之完全無眼球或虹膜缺失之迷你豬突變品種,故開發這樣的遺傳缺陷的迷你豬具有獨特性、新穎性、生醫與臨床研究之市場價值。
Abstract: Laboratory animal models commonly serve to mimic similar human pathology in biomedical and pathology researches. The results of the congenital or induced pathological changes using animal models could be used as references for the cause or effects of medical treatments of human diseases. A good animal model is the key for a successful medical research. Rodents are the most widely used animal model at present for human disease researches, however, the physiological, anatomical and genetic are not similar between rodents and human. Therefore, rodent model are not a ideal model for the study of particular human diseases. Pigs (sus scrofa) is an ideal experimental animal model because it offers a link between the classical rodent model and clinic human disease and application, the animal model of pig playing a key role in the development of new therapies and the improvement of existing medications in disease research.
Lanyu pig is an indigenous miniature pig of Taiwan. From 1975 to 1980, a program for the breeding and conservation for experimental animal (pigs) was promoted by the Taiwan Livestock Research Institute for the proposal “Development of pigs for biomedical research”. The Animal Science Department of National Taiwan University (Animal Science and Technology Department) bred Lanyu pig (female) and Landrace pig (male) to produce and selected white coated offspring produce the Lee-Sung strain pig (75% Lanyu pig, 25% Landrace, also as a miniature pig). In 2008, abnormal Lee-Sung pigs with anophthalmia and aniridia phenotype were discovered in the selected population, and the deficiency has been proved as a genetic defect.
Zebrafish, mice and rats have been used as animal models to study the genetic defects of the absence of the iris and eyeball. As the histological anatomy, physiology, life span and molecular genetics are quite different between rodents and humans, some studies concerning the defects in the development of eyeballs using animal models such as rodents could not be applied to humans. Previously induced mutation studies suggest that Six3, Rax, Lhx2, Pax6, Sox2 and Otx-2 were the aniridia and eyeless genes in the studies that were conducted in zebrafish and rodent. The eye defects are similar to the phenotype of the loss of iris and eyeball on the Lee-Sung strain, and the eye development defects of Lee-Sung strain pig are simply-inherited traits controlled by the aberrant expression of a few genes based on the results of our breeding study. Thus, we hypothesize that the anophthalmia in Lee-Sung strain pig may be caused by the deficiencies of Six3, Rax, Lhx2, Pax6, Sox2 and Otx-2 genes, and the aniridia may be due to the effect of impaired aberrant expression in those genes.
Notara et al. (2011) demonstrated that porcine is a suitable experimental model for studying the transplantation and the development of limbal epithelial cells through histology observation and transplantation approach. Therefore, it seems that using the anatomy, physiology and molecular genetic study of miniature pig deficient in eyeball genes could be a suitable medium and large animal model to understand eye development and therapeutic strategies for eye disease. We had completely obtained the DNA sequences information, including Pax6, Sox2 and Otx-2 genes from eye development deficit Lee-Sung pigs in 2014. In 2005, we intend to (1) establish a stable Lee-Sung pig purebred that produces the defective eyeball gene through breeding, (2) to set up the physiological and genetic data for anophthalmia, aniridia and wild-type Lee Sung pig animal mode. To sequence Six3, Rax and Lhx2, genes and considering the correlation between defective gene sequences and eyeball defect. (3) To confirm the histology of eye and the distribution of the cell layers for anophthalmia, aniridia and wild-type Lee Sung pig eye through immunofluorescence histology analysis (Histological section). Currently, there has not been a miniature pig breed with a whole eyeball defect mutation; hence, the defective genetic miniature pig is a unique, novel and valuable animal model for biomedical and clinical research.