以動物模式建立營養素或藥品影響骨骼發育的評估方法

To Establish an Animal Model to Evaluate the Effect of a Nutrient or Nutraceutical on Bone Growth

鄭秀卿(Hsiu-Ching Cheng)

指導教授 ： 蔡敬民

中原大學/理學院/化學研究所/碩士(2005年)

https://doi.org/10.6840/cycu200500749

摘要

本實驗旨在藉由鈣與大豆異黃酮對成長期大白鼠造骨細胞與蝕骨細胞活性之影響，欲建立可供檢測一保健食品或藥品對骨質密度影響的簡易可靠評估方法。實驗為期12週，採用3週大雌鼠與雄鼠，將56隻雌鼠隨機分成7組，依次餵予1/4Ca，1/2Ca，Ca (AIN-93G 鈣建議量) 或在上項三種飼料中另添加0.15% 大豆萃出物 (S：含0.027%異黃酮素) (1/4CaS，1/2CaS，CaS)，其中一組進行卵巢切除並給予CaS 飲食 (CaSOx)；另將24隻雄鼠隨機分成3組，分別餵予1/4CaS，1/2CaS，CaS飼料 (1/4CaS♂，1/2CaS♂，CaS♂)。實驗結束時測定血清中的骨質生化代謝指標，包括骨形成指標：骨源鹼性磷酸 (BAP) 與第一型膠原蛋白之碳端胜 (CICP)、骨回收指標：仳碄二酚胺 (Pyd) 與第一型膠原蛋白之氮端胜 (NTx)，並切片觀察骨組織型態學以計數造骨細胞、蝕骨細胞數目、骨質沉積速率、生長板厚度及進行骨小樑量檢測，另採用雙能量X光偵測儀分別測量股骨與脛骨骨密度。
實驗結果顯示，隨著鈣質攝取不足，血清BAP (p<0.05) 與CICP (p<0.01) 顯著降低，而Pyd顯著提高 (p<0.05)，但NTx則僅呈上升的趨勢 (p<0.10)。攝取添加大豆異黃酮飼料的動物，則其CICP比攝取同量鈣質的動物明顯上升 (p<0.01)、Pyd明顯下降 (p<0.05)，而BAP只有Ca組有明顯上升 (p<0.05)，NTx則無明顯變化。脛骨骨骼切片結果顯示，隨鈣劑量缺乏，造骨細胞數目、生長板厚度、骨量與鈣質沈積速率則隨之很明顯減少 (p<0.05)，但蝕骨細胞數目則很顯著提升 (p<0.05)；攝取添加大豆異黃酮之飼料可增加造骨細胞數目與骨量 (p<0.05)，鈣質沈積速率與生長板厚度則略有增加的趨勢 (p<0.10)，蝕骨細胞則沒有明顯變化 (p>0.10)。實驗結果顯示股骨密度隨著鈣質攝取量的下降而會很顯著地降低 (p<0.01)，脛骨密度亦成相同的之趨勢 (p<0.05)。攝取鈣量充足的動物，當多補充大豆異黃酮時則可再增高其骨密度 (p<0.05)。
本實驗結果發現，雄鼠的檢測結果其血液OB、OC活性指標大致與雌鼠相近。給予卵巢切除之雌鼠 (CaSOx) 與CaS組相比較，其血清CICP、BAP下降，而 Pyd、NTx 均上升，切片結果與骨密度測定皆呈現缺乏雌激素會增加骨質流失，並使生長板發育遲滯，降低骨密度。
由實驗結果亦可發現鈣質攝取量的增加或同時添加soy isoflavone，可提升成長期雌鼠與雄鼠之骨形成作用，同時亦具有抑制骨回收作用之生理功效，其亦能增加成長期鼠體內造骨細胞活性，並抑制蝕骨細胞活性，而isoflavone 與鈣質同時補充攝取，有加成之功效，能共同促進骨形成。
綜合上述結果，血清中的CICP、BAP及Pyd、造骨細胞數目、生長板厚度、骨量、以及鈣質沈積速率，均可作為反映生長期骨骼發育之骨密度變化的評估指標；而其中血清的CICP (最佳)，以及BAP和Pyd，均適宜作為檢測保健食品或藥品其影響骨骼發育功效程度的評估指標。

關鍵字

骨密度 ； 鈣質 ； 大豆異黃酮 ； 造骨細胞 ； 骨生化指標 ； 蝕骨細胞

並列摘要

The main goal of this study was to establish a simple and reliable method to evaluate the effects of a health food on growing bones. The study tried to use parameters of the effect of various levels of calcium (Ca) and soy extract (S) combinations on the activities of osteoblasts (OB) and osteoclasts (OC) in growing rats. Fifty-six 3 weeks old female Wistar rats were housed individually in stainless steel cages. Forty-eight of them were assigned into 6 dietary groups randomly, 8 each, and fed 1/4Ca, 1/2Ca, Ca (AIN-93G recommendation), or with 0.15% S (contained 0.027% isoflavone) in the diets (1/4CaS, 1/2CaS, CaS). Eight of them were ovariectomized and fed CaS diet (CaSOx). All rats were fed ad libitum for 12 weeks. After feeding the experimental diets, two bone formation markers, serum Bone-specific Alkaline Phosphatase (BAP) and C-terminal of type-I Collagen Propeptide (CICP), and 2 bone resorption markers, serum Pyridinoline (Pyd) and cross-linked N-telopeptides of type I collagen (NTx) were determined. Bone sections by histomorphometry were also employed to determine the numbers of OB and/or OC, trabecular Mineral Apposition Rates (MAR), the thicknesses of growth plate, and bone volume. Besides the above parameters, Bone Mineral Densities (BMD), a basic indicator of femurs and tibiae, were measured using Dual Energy X-ray Absorptometry (DEXA).
Results demonstrated that decreasing Ca intakes reduced bone formation markers, CICP (p<0.01) and BAP (p<0.05) and enhanced bone resorption marker, Pyd (p<0.05) significantly. Data revealed that supplement of soy extract highly elevated serum CICP (p<0.01) and BAP only with 1/4 Ca diets (p<0.05), however, suppressed the serum Pyd with 1/2 Ca diets. The bone sections also showed that calcium deficiency decreased OB numbers, thicknesses of growth plate, bone volume, MAR and increased OC numbers significantly, especially in CaS group comparing with others. Results also showed that supplement of soy extract could enhance the numbers of OB and the thicknesses of growth plate in tibiae (p<0.05). MAR showed a similar trend as BMD (p<0.05). Data demonstrated that increasing intake of Ca and/or soy extract enhanced the BMD of femurs and tibiae (p<0.05). It was also observed that ovariectomy significantly reduced the concentrations of serum CICP and BAP, and enhanced Pyd and NTx significantly (p<0.05) comparing with normal rats fed same diet (CaS). Both Ca and soy extract intakes could enhance bone growth and BMD in growing rats. Dietary supplement of soy extract can enhance Ca bioavailability and bone formation, and reduce bone resorption. Male rats got similar trend with females.
In conclusion, serum CICP, BAP, Pyd, OB numbers, thicknesses of growth plate, bone volume, MAR can be the bone biomarkers for growing bones. However, serum CICP (best), BAP and Pyd can be simple and sensitive markers to evaluate the effect of a heath food or nutraceutical on growing bones.

並列關鍵字

bone mineral density ； Isoflavone ； osteoclast ； calcium ； bone biomarker ； osteoblast

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