同種異體與異種骨髓間葉幹細胞於改善小鼠骨質疏鬆症之可能性

Abstract

全世界骨質疏鬆症患者人口於2006年底估計高達2億人，其中以老年人口及停經婦女患病率最高且有增加的趨勢。骨質疏鬆症主要成因為骨骼生成作用與骨骼溶蝕作用失去平衡，原本緻密的骨質變得空洞而脆弱，因而常導致多處骨折並引起併發症，嚴重者甚至造成生命的威脅，於是在老齡化社會體系中，建立如何預防及治療此疾病之模式已成為現今重要之課題。 鑒於骨質疏鬆症病人之間葉幹細胞分化成造骨細胞的能力已受損，因此本試驗的目的為建立骨質疏鬆症小鼠與探討骨髓間葉幹細胞治療此疾病的可能性。將15週齡之C57/BL6小鼠摘除卵巢6個月後，經由病理組織切片染色可檢測出生長板上方與下方骨小樑之密度明顯地下降，微電腦斷層掃描檢測顯示正常小鼠與骨質疏鬆症小鼠骨質密度百分比為: 15.00 ± 1.99%相對於9.70 ± 1.44% (P<0.05)，顯示這些小鼠已產生嚴重的骨質疏鬆症。 另外，由本實驗室產製之綠色螢光轉基因鼠與豬分別分離純化其間葉幹細胞，經骨分化培養液 (0.1μM dexamethasone + 10 mM glycerol-2-phosphate + 50μM ascorbate-2-phosphate) 處理後，以BCIP/NBT與Alizarin Red S 染色法證實分化後的間葉幹細胞確實具有骨細胞的特徵與功能。此後，將同種異體與異種間葉幹細胞以尾靜脈注射或腹腔注射的方式，分別注入尾靜脈注射組7隻與腹腔注射組5隻骨質疏鬆症小鼠體內進行觀察。該間葉幹細胞因表現水母綠色螢光蛋白質而可追蹤其在小鼠體內之功能與分化潛能。綠色螢光小鼠或豬骨髓間葉幹細胞之遷移與參與骨生成之潛能則由免疫組織化學染色法、骨頭病理切片與微電腦斷層掃描偵測。試驗結果顯示: 同種異體尾靜脈注射組，5隻試驗小鼠中有2隻骨質密度增加，其骨質密度增高比率為35.09%與19.32%；同種異體腹腔注射組，5隻試驗小鼠中有1隻骨質密度增加，其骨質密度增高比率為18.59%；異種尾靜脈注射組，6隻試驗小鼠中有2隻骨質密度改善，其骨質密度增高比例為13.27%與7.98%；異種腹腔注射組，5隻試驗小鼠中有1隻骨質密度改善，其骨質密度增高比率為39.38%。除此之外，在受試骨質疏鬆症小鼠之骨髓中，可測得注入之間葉幹細胞的存在，推測可作為骨前身細胞來源之儲存庫，且能作為未來之骨修復。 綜合上述，同種異體或異種之骨髓間葉幹細胞不論經由尾靜脈注射或是腹腔注射之方式，藉由免疫組織化學染色法皆可發現遷移至股骨中，經過骨頭病理切片與微電腦斷層掃描偵測出有新形成之骨小樑。因此，應用同種異體或異種成體骨髓間葉幹細胞於骨質疏鬆症治療，在未來具臨床治療應用之可能性。

It was estimated that patients suffered from osteoporosis worldwide were exceeding 200 millions in the year of 2006, and still increasing prevalently in the elderly and menopausal women. This illness, which causes serious bone fractures, leads to life threatening complications and greatly influences the quality of patients’ life. Osteoporosis is a result of imbalance between bone formation and resorption and gradually become a disease needs to be resolved urgently in the modern society. Osteoporosis is due to defect in mesenchymal stem cells (MSCs) that leads to reduce the ability to differentiate into osteoblast. For this reason, the objective of this study was to establish a mouse model of osteoporosis and investigate the feasibility of using allogenic or xenogenic bone marrow mesenchymal stem cells (BMMSCs) as therapeutic agents. C57/BL6 female mice at fifteen weeks of age were operated for ovariectomy to induce osteoporosis. Assays of bone sections and micro computed tomography (μCT) scan were performed to examine the bone density percentages on those animals six months post the ovariectomy. Bone sections could reveal the density of trabecular bone clearly dropped above and below the growth plate. μCT scan also showed bone density significant decreases: 15.0 ± 1.99% versus 9.7 ± 1.44% (P<0.05) indicating these mice has become osteoporosis. In addition, bone marrow derived MSCs obtained from transgenic mice and pigs harboring the enhanced fluorescent protein (EGFP) were treated with differentiating solution (0.1μM dexamethasone + 10 mM glycerol-2-phosphate + 50μM ascorbate-2-phosphate) and analyzed by BCIP/NBT and Alizarin Red S stains to confirm the potential of osteogenesis prior to the allogenic or xenogenic stem cells transplantations either through intravenously (IV) or intraperitoneally (IP), IV group has 7 osteoporotic mice and IP group has 5 osteoporotic mice for experiment respectively. The EGFP reporter was later used for tracing the migration fate of EGFP-m/pBMMSCs after they had been transplanted into osteoporotic mice, and also for further verifying those functions and differential potential of transplanted cells in vivo. The ability of migration and involvement of bone formation for EGFP-m/pBMMSCs were detected in the immunohistochemistry, bone section and and μCT scan. Increased bone density were observed in two out of five in allogenic IV: 35.09%、19.32%, one out of five in allogenic IP: 18.59%, two out of six in xenogenic IV: 13.27%、7.98% and one out five in xenogenic IP: 39.38%. Besides, some EGFP-m/pBMMSCs were found to remain in the bone marrow and might serve as a reservoir of bone progenitors which could be used for bone repairing later. The results indicated that EGFP-m/pBMMSCs were injected into osteoporotic mice by IV or IP were able to migrate into the bones and participate in the bone formation in vivo, display histologically the location of EGFP-m/pBMMSCs in bone, examined by GFP immuno-staining. Assay of pathogenic histology and μCT scan illustrated improved microstructures in newly formed trabecular bone tissue. Hence, allogenic or xenogenic BMMSCs possess the feasibility to be as a clinical therapeutic agent with an improved curative efficiency for treating osteoporosis in the foreseeable future.