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研究生:呂宜鄉
研究生(外文):Yi-Hsiang Lu
論文名稱:環境雌激素樣作用物質對骨細胞分化影響
論文名稱(外文):Effects of environmental estrogens on the osteoblast and osteoclast differentiation
指導教授:劉興華劉興華引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:毒理學研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:66
中文關鍵詞:環境雌激素造骨細胞分化破骨細胞分化薏苡仁水萃取物苯駢芘三丁基錫
外文關鍵詞:environmental estrogenosteoblastosteoclastadlay extractbenzo(a)pyrenetributyltin
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骨骼是具有數種細胞型態的複雜組織,而其中最重要的兩種分別是負責骨吸收作用的破骨細胞及職司骨生成的造骨細胞。破骨細胞具有多核的細胞型態來自於血液性幹細胞的分化,其前趨物存在於單核球╱巨噬細胞系列;而造骨細胞則是從骨髓體細胞中的間葉性幹細胞分化形成。當骨骼進行再造作用時,破骨細胞會經由分泌酸性物質或一些蛋白酵素去移除老化或受損的骨組織,接著破骨細胞會移動到骨組織內進行骨吸收作用然後凋亡,之後造骨細胞會形成新的基質骨細胞並在鈣化後成為新的成熟的骨骼組織;而骨質疏鬆症發生的原因即在於骨吸收和骨生成作用失去平衡,也就是體內造骨細胞和破骨細胞間交互作用的平衡失去了。
環境荷爾蒙,包括雌激素與雌激素樣物質,其作用如同內分泌干擾素,一旦環境荷爾蒙經由環境媒介進入生物體內,會因為具有類似生物體內荷爾蒙之功能,而干擾生物體內的代謝、合成、細胞分裂及再生的作用,此外,在對人體健康方面可能的影響包括:發育不良、智力減少、生育力降低、神經傷害,甚至影響免疫系統的正常功能。
在本實驗中,我們觀察三種環境雌激素樣物質:薏苡仁水萃取物、苯駢芘(benzo(a)pyrene)及三丁基錫(tributyltin)對造骨細胞和破骨細胞分化的影響。薏苡仁屬於草本植物,已經被使用在中藥方面且也是一種食物,具有抗過敏、抗突變及降血脂的能力;苯駢芘是已知會提升氧化壓力具有致癌及致突變性的環境污染物;而三丁基錫是被廣泛添加在抗海洋生物的油漆、農業的殺蟲劑及塑膠穩定劑裡的成分,其在近年對海洋水生環境造成了相當大的污染問題。在我們的實驗中,我們使用前造骨細胞 MC3T3E1做細胞培養、血液性幹細胞與 MC3T3E1的共同細胞培養和去卵巢小鼠模式,去觀察這三種物質對骨細胞分化的影響。
實驗的結果顯示,苯駢芘在長時間跟短時間的暴露均會增加 COX2和pp38的蛋白質表現,這兩種蛋白已有很多期刊報告說對造骨細胞的分化是必須的,但是在鹼性磷酸酶及骨礦物質化染色中卻呈現出相反的反應。薏苡仁水萃取物在共同細胞培養方面可增加造骨細胞的分化能力,同時破骨細胞分化的能力也上升了;對於去卵巢小鼠的觀察,不管是骨骺組織或是其組織培養在骨密度、鹼性磷酸酶和鈣質都呈現下降的反應,而酸性磷酸酶的活性會上升,但是在給予含薏苡仁的飼料或直接以餵管餵食薏苡仁萃取物,都可以讓骨流失情形緩解。至於三丁基錫,共同細胞培養顯示對造骨細胞及破骨細胞的分化上並無有意義的變化。
所以依據我們所做出的實驗結果,雖然苯駢芘會增加COX2和pp38的蛋白表現,但造骨細胞的分化作用仍下降,但若同時添加NS398,則分化能力有恢復現象,這或許可推測說COX2活性在抑制造骨細胞分化上扮演了一個重要角色;薏苡仁在共同細胞培養可以增加造骨細胞的分化作用,且對骨質疏鬆狀態的小鼠具有緩解的能力,因此或許是能促進造骨細胞分化的營養食品;至於三丁基錫由於實驗的數據不多,推測其或許並不影響造骨細胞和破骨細胞的分化作用。
Bone is a complex tissue composed of several cell types and the two major of them responsible for bone remodelling are osteoclast, which resorb bone, and osteoblast, which form new bone. Osteoclasts are multinucleated cells of haemopoietic origin (HSCs), which differentiate from precursors in the monocyte/macrophage lineage. And osteoblasts are cells of mesenchymal origin (MSCs) which differentiate from bone marrow stromal cells. During the bone remodeling cycle, old or damaged bone is removed by osteoclast, by the secretion of acid and proteolytic enzymes onto the bone surface. Subsequently the osteoclasts migrate away from the area of bone undergoing resorption and undergo apoptosis. They are replaced by osteoblast, which lay down new bone matrix in the form of osteoid. Later, the osteoid becomes calcified to form mature bone. And osteoporosis is a reduction in skeletal mass due to an imbalance between bone resorption and bone formation, whereas bone homeostasis requires balanced interactions between osteoblast and osteoclast.
Environmental hormones acting as endocrine disrupters include oestrogens and oestrogen-like products. Once environmental hormones enter an organism via an environmental medium, if may interfere with the metabolism, synthesis, cell division and reproduction functions of the organism. Some effects on human health may include, dysplasia, intelligence reduction, infertility, nerve damage, increasing in violent and severe diseases such as disorders of the immune system.
In this study, we investigated the expression of osteoblast differentiation and osteoclast differenation by adlay extract, benzo(a)pyrene (BaP) and tributyltin (TBT), three environmental estogens. Adlay is a grass crop which has been used in traditional Chinese medicine and also as a nourishing food. It has been shown to posses anti-allergic, antimutagenic and hypolipemic effects. BaP is a known environmental pollutant which causes oxidative stress, carcinogenic and mutagenic compound. TBT is widely used as antifouling paints, agriculture biocides, and plastic stabilizers around the world, resulting in great pollution problem in aquatic environments. In our studies we measured the effects of these three materials on the MC3T3E1 (preosteoblast cell line) cells, co- culture of MC3T3E1 and HSCs and ovariectomic (Ovx) mouse model.
The results shown that BaP induced the expression of COX2 and pp38 protein, which have reported are necessarily for differentiation in osteoblast cells. However, the ALP activities and mineralized staining had negative responses and reversed when combined with NS398. Adlay extract, in the co-culture system, could increase the differentiation of osteoblast, while the differentiation of osteoclast also was enhanced. In Ovx mice, the levels of BMD, ALP and calcium in bones were inhibited, while the increased ACP activities in metaphyseal tissues were shown. Treatment with adlay (diet or extracts) could reverse those effects in ovx mice. TBT, in co-culture system, could find significant changes neither in osteoblast nor in osteoclast differentiation.
According to our results, since BaP increased the expression of COX2 and pp38 to reduce the osteoblast differentiation and this phenomenon can reverse by treatment of NS398, but not by treatment of SB203580, suggesting that COX2 activation plays an important role on osteoblast differentiation. Adlay could increase osteoblast differentiation, in co-culture system, and reverse the osteoporotic status in mice, would be a helpful healthy food for enhance osteoblast differentiation. However, about, TBT, we didn’t have enough data to support its effect on the differentiation of osteoblast or osteoclast.
中文摘要………………………………1
英文摘要………………………………4
緒論………………………………7
材料與方法……………………………… 15
實驗結果………………………………29
討論………………………………35
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