應用卵巢摘除術建立骨質疏鬆症豬模式可行性之探討

Abstract

骨質疏鬆症為目前最為常見之骨骼相關疾病，根據世界衛生組織之統計，全世界年齡超過65歲之婦女，有35%受到此疾病所苦。雖然骨質疏鬆症是如此常見之疾病，但時至今日仍沒有一既安全且有效之相關療法被成功開發推出。目前較常見之激素療法，因有可能提高乳癌之罹患率而少被採用。根據統計資料顯示，美國每年花費在骨質疏鬆症病患之療照養費用，可高達100至150億美金之譜，顯示骨質疏鬆症不僅對人類健康有相當之危害，遂對經濟與社會成本方面也會造成極大之壓力。故深入研究骨質疏鬆症進而開發有效之療法乃成為一項極重要之課題。設若能成功建立良好之模式動物，模擬人類生理與解剖結構之骨質疏鬆症動物模式，則能於相關之研究提供諸多助力。 賓朗豬因其體形之小型化及其白色之皮膚毛髮而被認為甚適合提供做為試驗動物模式之使用。本研究旨在探討以卵巢摘除在賓朗豬身上建立骨質疏鬆症動物模式之可能性。試驗之第一部分為骨質疏鬆動物模式之建立，選用10月齡的賓朗豬9頭，以外科手術移除卵巢的方式模擬婦女停經，以此方式建立第一型骨質疏鬆症之動物模式。在卵巢移除的16個月後對其骨質密度進行分析並與3隻控制組豬隻進行比較，以檢視此模式建立之成效，因豬之骨質相關資料庫參數不足造成試驗組與控制組之個體分析數值無法精確轉換，而導致比較失去意義。為了取得賓朗豬之骨髓間葉幹細胞進行分析，並評估對其進行細胞移植之可能性，第二部分試驗即在於賓朗豬之骨髓間葉幹細胞之分離與純化，結果顯示賓朗豬骨髓間葉幹細胞除符合間葉幹細胞之特性外，其表現特徵與洋豬骨髓間葉幹細胞也具有高度相似性。第三部分之試驗則是以骨髓間葉幹細胞移植的方式，重新驗證此骨質疏鬆症動物模式建立之成效，結果顯示在經卵巢移除17個月後再接受間葉幹細胞移植之豬隻其骨質密度與控制組並無顯著差異，顯示卵巢摘除並未於豬隻身上造成顯著之骨質流失。最後一部分之試驗為卵巢摘除豬之間葉幹細胞與健康豬間葉幹細胞之增殖與分化能力比較，結果顯示其兩種來源細胞在聚落形成能力、增殖速度與硬骨分化能力上皆無顯著差異。 綜合上述，卵巢摘除17月並未能於賓朗豬身上造成顯著之骨質流失，也未對其骨髓間葉幹細胞之增殖與分化能力造成顯著之影響，顯示卵巢摘除之方式似乎不適合用於以賓朗豬建立骨質疏鬆症之試驗動物模式。

Osteoporosis is one of the most common bone disease, according to World Health Organization, 35% of women above 65 year old are suffer for this disease. Although osteoporosis is such a common disease, there is not a safe and effective treatment so far, the commonly used hormone treatment is gradually abandoned because it may raise the potential of breast cancer. On the other hand, America spend ten to fifteen billion dollars on osteoporosis patients every year, so to develop a new effective treatment is very urgent, by that, if we can establish an animal model that simulate human physiology and anatomy better, then the development of osteoporosis treatment would be much easier. To study the possibility of using ovariectomy as method to establish a porcine osteoporosis model, Binlang pigs were chosen for experimental candidates, BL pig has the same genetic composition as Lanyu pig but white-skinned, and its small size and white skin make it better for clinical model animal. First experiment was to establishment osteoporosis model of BL pig, nine BL pigs aged ten months were ovariectomied for simulating the menopause of women and develop type 1 osteoporosis. Sixteen months after ovariectomy, dual-energy X-ray absorptiometry (DEXA) were used to measure the bone marrow density (BMD), and compare it with the control pigs to verify the establishment of this model, but the database size is too limited makes the data tough to convert precisely. Secondly, BL pig’s bone marrow-derived mesenchymal stem cells (MSCs) were isolated, tri-linage differentiation and surface antigen detection were conducted to confirm the characteristics of MSC, and verify the possibility of MSC transplantation. In the third experiment, MSC were transplanted to the 6 out of 9 ovariectomied pigs to verify the establishment of this model, and this approach as a treatment for osteoporosis. The results of BMD score scan by DEXA and micro-CT show that have no significant difference (p > 0.05), 2 and 3 months after cell transplantation, respectively. The last experiment is to compare the proliferation and osteogenesis capacity of MSCs isolated from marrow fluid of ovariectomied pigs and healthy Ds-red transgenic pigs. The results reveal that they have no significant difference on colony forming rate, doubling time and osteocyte differentiate ability. In conclusion, there is not significant bone loss happened on BL pigs ovariectomied for 17 months, and shows no significant effects on Binlang pig bone marrow derived MSC’s proliferation and osteogenic ability. It seems that ovariectomy might not suitable to establish osteoporosis model of BL pig, another ways are worth to consider for establishing pig model of osteoporosis.