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Title: | 中草藥應用於關節軟骨組織工程之研究 The Effects of Chinese Herbal Medicine on Chondrocytes for Cartilage Tissue Engineering |
Authors: | Wen-Yang Lin 林文央 |
Advisor: | 林峰輝 |
Keyword: | 中草藥,軟骨,組織工程,定量聚合酵素鏈鎖反應,自由基,抗氧化, Chinese herbal medicine,Cartilage,Tissue Engineering,Real-time RT-PCR,ROS,Aucubin,Betulin, |
Publication Year : | 2007 |
Degree: | 碩士 |
Abstract: | 人類的骨骼是由硬骨和軟骨構成的器官,具有支持、保護和運動的功能。軟骨是由僅佔總體積百分之五的軟骨細胞和其所分泌的大量細胞外基質組成,在健康的軟骨內,其細胞外基質主要為第二型膠原蛋白和多醣類,由於它們攜帶大量的負電性,使水分子及正電離子易受吸引而聚集到軟骨中,因而使軟骨在受到壓力時,能具有吸收震動力而達緩衝的效果。
然而受到破壞的軟骨其自我修復的能力很有限,除了因為軟骨中沒有血管或神經的分布,使得受傷的部位沒有血流侵入而攜帶發炎因子,因而無法啟動修復機制;另外在軟骨中的軟骨細胞數量本來就很有限,即使受傷部位鄰近的細胞可以進行細胞分裂,但卻因數量過少且加上黏滯的細胞基質阻礙了細胞的遷移;而當受傷侵入軟骨下方的硬骨時,受到破壞的硬骨內的血流便有機會侵入軟骨而啟動修復,但此時新生成的軟骨組織,只具有分泌第一型膠原蛋白細胞外基質的能力,而這種第一型膠原蛋白的機械特性和強度與原本第二型膠原蛋白相差甚大,最終也將被逐漸降解而喪失軟骨的功能。 於是受到創傷而無法自我修復的軟骨會漸漸影響到整個關節,轉變為退化性骨關節炎。目前臨床上的藥物療法皆著重在止痛而無法有根治之效,最常用的藥物有普拿疼、第二型環氧脢抑制劑、非類固醇類抗發炎藥物、關節內注射透明質酸、關節內注射類固醇,然而這些只治標不治本的療法,最終只能藉由置換人工替代膝關節來取代壞損的關節。 利用組織工程在體外培養軟骨組織近年來已成為修補受損關節軟骨的途徑之一,組織工程利用組織工程的三要素:細胞、生物材料骨架及訊號的刺激,模擬組織在體內的狀態,以期在體外復育仿生組織。此研究從具有促進軟骨再生之效的傳統中草藥複方中,萃取出主要來自於車前子及山葡萄兩種藥材中的兩種主要有效單一化學成分,並以適當濃度添加到二維及三維支架的軟骨組織培養系統中,而後利用同步定量聚合酵素鏈鎖反應、細胞總DNA、細胞外基質葡萄胺聚醣濃度、及免疫螢光染色確定細胞增殖情形;並利用化學發光技術配合適當的探針,偵測軟骨細胞中自由基的產生量及其抗氧化的能力。 實驗結果發現,二維單層軟骨細胞的培養中,利用適當濃度的車前子或山葡萄有效單一化學成分之添加,軟骨細胞能夠增加其細胞外基質相關的基因表現,並能抑制與細胞外基質降解相關之蛋白質的基因表現;也由二維系統初步發現,由山葡萄萃取之有效單一化學成分相較於由車前子萃取之有效單一化學成分,更具活性氧之清除能力,因而使得軟骨細胞的整體活性在山葡萄萃取物之添加組更為提升。在三維軟骨組織工程的培養中,經過兩週以上培養的軟骨細胞明顯地增加細胞活性及複製能力,其細胞外基質相關的基因表現也明顯的上昇,而與細胞外基質降解相關的基因被受到抑制;值得注意的是,經過四週以上培養的軟骨細胞,其細胞整體活性相較於一週及兩週更為佳,或許建議在未來之臨床應用也宜取經過四週培養之軟骨組織工程物為較佳。 The skeleton is composed of cartilage and bone which provide the functions such as support, protection, and movement in daily life. Articular cartilage tissue is composed of chondrocytes and extracellular matrix, where the chondrocytes only make up less than 10% of the total volume of cartilage. In healthy cartilage, the matrix is composed of collagens, especially type II collagen, proteoglycans, and noncollagenous proteins, and is filled with water because of the hydrophilic property of the framework. Cartilage possesses limited ability to achieve spontaneous repair due to its dense extracellular matrix and lacking of blood vessels, lymphatics and innervation. When lesions occur in articular cartilage, there is no bleeding and thus no mechanism for the replacement of lost or damaged tissue. Neighboring chondrocytes may respond by local proliferation; however, because they are sequestered in the dense matrix, they do not migrate into the damaged region to fill the void. If injury extends through the chondral layer to the subchondral bone and underlying vasculature, a repair response can hence occur, but the newly formed cartilage will be gradually populated with type I collagen and degenerate to a fibrocartilaginous scar tissue after 6–8 months. Circumstances that impair chondrocytes function hence disrupt the balance of synthesis and catabolism and lead to the development of osteoarthritis. In the progression, osteoarthritis eventually impairs the function of a whole joint, including the cartilage, the subchondral bone, the synovium and the periarticular connective and muscular tissues. The current treatment options are fairly limited which include only symptomatic treatment of limited efficacy with analgesics, non-steroidal anti-inflammatory agents or intra-articular administration of steroids or hyaluronic acid, and if the joint destruction can not be halted, the ultimate measure is joint replacement. Tissue engineering has been a feasible way to regenerate cartilage in vitro, which combines cells, scaffolds, and signals to mimic the original environment of tissues in vitro. In addition, there are some Chinese herbal medicines have been used to treat the degeneration of the cartilage for thousands of years, but the precise mechanism of their potent chondrogenesis effects have not been fully elucidated. Therefore, in this study, we investigated the involved mechanism of two single chemical compounds: aucubin and betulin which were separately extracted from Plantago asiatic and Ampelopsis brevipedunculata (Maxim.) Trautv. by focusing on their proliferation and antioxidant activity. The experiment was divided into two parts. One was two-dimensional chondrocytes culture; the other was three-dimensional scaffold culture. After treating two-dimensional chondrocytes or three-dimensional seeded chondrocytes with optimal concentration of single chemical compounds, the proliferation and matrix productivity of them were evaluated by real-time reverse-transcriptase polymerase chain reaction, ELISA assays, and immuno-histochemical staining. The important role in scavenging free radicals by those extracted chemical compounds was detected by the chemiluminescence method. The results showed that, in two-dimensional chondrocytes culture, both aucubin and betulin could effectively promote the mRNA expression of ECM and inhibit the mRNA expression related with ECM degradation at appropriate concentration, and the ability of O2•‾ scavenging made aucubin and betulin as protectants of chondrocytes, which would stimulate chondrocyte proliferation and maintain the basic chondrocyte activities. In three-dimensional scaffold culture environment, betulin can significantly stimulate chondrocyte proliferation and maintain the basic chondrocyte activities until four-week cultivation, which suggested that in the future application the in vitro three-dimensional cultivation of chondrocyte-scaffold hybrids should be maintained more than four weeks, and of course the addition of 0.32μg/ml of betulin into the cultured environment possessed positive effects toward chondrocytes and the whole cartilage-mimic tissue. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/9943 |
Fulltext Rights: | 同意授權(全球公開) |
Appears in Collections: | 醫學工程學研究所 |
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