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研究生:蕭雅文
研究生(外文):Ya-Wen Hsiao
論文名稱:紫蘇籽油可改善切除卵巢之大鼠所造成的骨質疏鬆症
論文名稱(外文):Perilla Seed Oil Ameliorate Osteoporosis in Ovariectomic Rat
指導教授:李彗禎
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生化暨生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:92
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缺乏雌激素會增加體內的氧化壓力並導致骨質疏鬆。在多種植物油中,紫蘇 (Perilla frutescens, PO) 籽油的ω-3含量為最高約54~64% ,並且會代謝成EPA和DHA。亦有文獻證實EPA和DHA有很好的抗氧化活性,文獻證實在氧化壓力的環境會抑制成骨細胞的分化和增生,本實驗觀察紫蘇籽油是否可以改善骨質疏鬆症。將SD大鼠分為六組,其中一組做偽手術當作控制組,將二十隻老鼠做卵巢切除手術,並分為OVX與每天餵食紫蘇籽油(0.1%、0.5%、1%),八週後取血液生化值與股骨觀察其骨小樑結構與蛋白表現量。研究結果發現卵巢切除大鼠的大腿骨內脂肪和成骨細胞增加而骨小樑密度降低,在餵食紫蘇籽油後明顯改善。OVX大鼠血中鹼性磷酸酶 (ALP) 濃度明顯增加,給予PO後可有效降低ALP 值。結果顯示卵巢切除之大鼠餵食PO後氧化型低密度脂蛋白比起正常組別也有明顯減少。在我們的研究發現OVX之母鼠Runx2明顯降低,餵食PO後有回復情形。缺乏卵巢之母鼠的RANK與RANKL增加,而餵食PO後降低其表現量。此外,我們在細胞模式中探討PO是否可保護MC3T3-E1免於氧化型LDL誘導氧化壓力所造成的傷害。將MC3T3-E1 細胞培養於含有10μg LDL 和10Μm (CuSO4) 的骨誘導培養液中,並添加不同濃度的PO,進而檢測成骨細胞存活及氧化損傷的指標。氧化型LDL降低ALP 活性明顯低於單純誘導分化組。PO可增加 ALP 的表現及活性。我們的研究指出紫蘇籽油可預防停經症候群所造成的骨質疏鬆症。

Estrogen deficiency can increase oxidative stress and cause osteoporosis. In comparing to other plant oils, perilla seed oil (PO) consistently contains higher proportion of ω-3 (ALA) fatty acids at 54–64%, and it metabolized into eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6). The previous studies showed EPA/DHA possessed a good antioxidant activity. This study aims to explore whether PO can improve osteoporosis. Furthermore, it is determined whether PO could protect osteoblasts from oxLDL induced oxidative stress in the MC3T3-E1 cells. Female Sprague-Dawley rats were divided into 6 experimental groups, five days after ovariectomy, 20 ovariectomized (OVX) rats were divided into 4 groups, randomized to treat with PO (0.1%, 0.5%, 1%) oral gauge daily for 8 weeks, sham were used as controls. At the end of treatment serum biochemical parameters measure. Femurs were removed and tested for trabecular micro-architecture and protein expression. MC3T3-E1 cells were cultured in the osteogenic medium with or without 10μg/mL and 10mM (CuSO4) or different concentrations of PO. The results show the ratio of adipocyte/osteoblast increase and trabecular bone density decrease dominantly in femur bone of ovariectomic rat, and perilla seed oil ameliorates the status significantly. Previous studies demonstrate oxidative protein products inhibit the proliferation and differentiation of rat osteoblasts via oxidative stress. In this sduty, the expression of oxLDL decreased after feed PO in ovariectomic rat. The expression of Heme oxygenase-1 (HO-1) shows the oxidative status in individual. Herein, HO-1 is decreased in bone tissue of ovariectomic rat, but it is restored after treating with PO. RANK and RANKL are important components to regulate osteoclast proliferation. The expression of RANK/RANKL increased in femur bone of ovariectomic rat, and perilla seed oil can reduce the expression of RANK/RANKL. MC3T3-E1 cells were cultured in the osteogenic medium with or without 10μg and 10Μm (CuSO4) or different concentrations of PO, and markers of osteoblast function and oxidative stress were examined. As compared with differentiation incubation, ALP activity decreased after treating with oxLDL, and PO can increase the expression of ALP activity in osteoblastic MC3T3-E1 cells under the presence of oxLDL. Taken together, the results show that perilla seed oil has the potential to prevent women from osteoporosis in postmenopausal syndrome.

縮寫表 1
中文摘要 3
英文摘要 5
第一章、 緒論 6
一、 骨骼組織: 7
1-1. 骨骼 7
1-2. 骨骼中主要細胞 8
二、 骨生成 (osteogenic) 9
2-1、 成骨細胞生長、分化與作用 10
2-2、 OB胞外基質 12
2-3、 OB生長分化過程中基因表現的變化 13
2-4、 調控蝕骨細胞新生生作用 (osteoclastgenesis) 14
2-5、 RANKL/RANK分子機制: 14
2-6、 ITAMs 分子機制: 16
三、 氧化壓力 16
3-1、 活性氧族 (reactive oxygen species;ROS) 17
3-2、 活性氮族 (reactive nitrogen species;RNS) 17
3-3、 酵素抗氧化系統 (enzymatic antioxidant) 19
3-4、 非酵素抗氧化系統 (non-enzymatic antioxidant) 19
四、 骨質疏鬆症 (osteoporosis) 21
4-1、 「原發性」: 又稱退化性骨質疏鬆症,可分為 22
4-2、 「繼發性」: 23
4-3、 骨質疏鬆的診斷: 24
4-4、 骨質疏鬆的預防: 25
4-5、 骨質疏鬆的治療: 26
五、 低密度脂蛋白 (low density lipoprotein;LDL) 27
5-1、 基本介紹 28
5-2、 LDL的氧化 29
六、紫蘇籽油 30
6-1、 成分與應用 30
第二章、 研究動機 33
第三章、 實驗設計 34
第四章、 實驗儀器 36
第一部分、探討紫蘇籽油對卵巢切除之大鼠所引起的骨質疏鬆症作用 39
1-1、 蘇籽油(PO)取得 39
1-2、 動物來源與骨質疏鬆症動物誘導 39
1-3、 血液生化值檢測 40
1-4、 石蠟組織包埋及切片 42
1-5、 免疫組織染色 (Immunohistochemistry) 44
第二部分:探討紫蘇籽油對oxLDL引起氧化壓力抑制前造骨母細胞分化的影響 46
2-1、 細胞來源及培養 46
2-2、 細胞模式建立及方法 47
2-3、 Conditioned Medium (CM) 47
2-4、 Cell proliferation assay (MTT assay) 47
2-5、 低密度脂蛋白 (LDL) 的分離 48
2-6、 TBARs assay 48
2-7、 Alkaline phosphatase activity assay (ALP activity assay) 49
2-8、 Alkaline phosphatase stain (ALP stain) 50
第三部分:實驗溶液配製 53
免疫組織染色配製: 53
1× PBS-T 配製 54
SIGMA FAST pNPP substrate solution 配製: 54
第四部分:數據分析 54
第七章、 實驗結果 55
一、動物實驗結果 55
1-1、 處理PO對骨質疏鬆大鼠體重之影響 55
1-2、 處理PO 對骨質疏鬆大鼠血液生化值之影響 55
1-3、 處理PO對骨質疏鬆大鼠骨結構之影響 56
1-4、 PO增加骨質疏鬆大鼠骨中第一型膠原蛋白之表現 56
1-5、 PO 骨質疏鬆大鼠骨中oxLDL之表現 56
1-6、 PO增加骨質疏鬆大鼠骨中Runx2 之表現 57
1-7、 PO降低骨質疏鬆大鼠骨中HO-1 之表現 57
1-8、 PO 降低骨質疏鬆大鼠骨中 RANKL 之表現 58
1-9、 PO 降骨質疏鬆大鼠骨中RANK 之表現 58
二、 細胞實驗結果 59
2-1、 氧化型LDL對於MC3T3-E1毒性試驗 59
2-2、 PO 對氧化型LDL處理MC3T3-E1 生長之影響 59
2-3、 PO對氧化型LDL活性測試 60
2-4、 氧化型LDL誘導下 PO對對鹼性磷酸酶活性之影響 60
2-5、 PO在氧化壓力條件下對ALP表現形態之影響 61
2-6、 PO在氧化壓力條件下成骨細胞分化蛋白表現之影響 61
第八章、 討論 63
一、 PO對骨質疏鬆症大鼠生理之影響 63
1-1. PO 對骨質疏鬆症大鼠體重之影響 63
二、 PO 對停經引發骨質疏鬆症之影響 64
2-1、 PO 對骨質疏鬆大鼠血中ALP 及骨結構之影響 64
2-2、 PO對骨質疏鬆症大鼠OB 之影響 64
三、 PO 對OB 骨生成作用之影響 64
3-1、 PO對於MC3T3-E1 在生長方面之影響 64
3-2、 PO對於MC3T3-E1 在分化方面之影響 65
第九章、 參考文獻 67
第十章、 實驗結果圖表 75
第十一章、 附錄 90



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