3-D 培養模式下脫氫表雄酮對軟骨細胞的影響
Effects of dehydroepiandrosterone on chondrocytes in a three-dimensional culture model
中文摘要
隨著年紀的增加、生理性關節活動變小或重力的壓迫，例如肥胖會使軟骨退化、磨損、關節表面 凹凸不平，嚴重時軟骨會完全喪失，骨頭與骨頭直接接觸會導致發炎、紅腫、疼痛，甚至無法正 常彎曲、移動。由於身體的老化過程，不能再生產足夠的蛋白多醣和合成膠質來維持健康的軟骨 結構，老化的關節愈來愈沒有彈性，而造成關節軟骨的磨損。脫氫表雄酮
Dehydroepiandrosterone (DHEA)是一種人體重要的荷爾蒙，DHEA 是性荷爾蒙的前驅物，
由腦部、皮膚和腎上腺所製造，也可由山藥、芋類植物或鹿茸中萃取得到，在體內會轉變成為雄 性素與雌性素，這兩種激素在人體中對於骨的維持非常地重要，所以可能因此而對軟骨有幫助。
本研究欲探討在軟骨受傷害的情況下，是否有治療或保護的作用，更進一步的說，或許可提供另 一個對於軟骨傷害治療的新方向。
本實驗取用初生四天之幼鼠為動物模式，取其膝關節軟骨培養軟骨母細胞，並採用三度空間培養 法 (3-dimensional cultures) 培養軟骨細胞，然後在加入 Dehydroepiandrosterone (DHEA) 或在 LPS 與 SNAP 分別來模擬細菌性關節炎與自然老化退化性關節炎的情形，並檢測 軟骨細胞增生情形，再進一步的檢測軟骨細胞內基質 glycosaminoglycans (GAG)，使用 DMB assay，而 total collagen 量的多少，則使用 OHP assay 及一些會造成軟骨傷害的物質的表現，
如 nitric oxide (NO)、prostgalends (PGE2)、Interleukin-6 (IL-6)、metalloproteinases (MMPs)。並且會探討 MMPs 與 TIMP-1 的表現，與即時定量反轉錄聚合?連鎖反應來探討基因 COX-2、iNOS、MMP1、MMP3、MMP13。
實驗結果顯示，DHEA 在誘導軟骨細胞增生時，在 10-6M 濃度下時最好，在加有 H2O2 情況 時，在 10-6M 濃度下 DHEA 對自由基 H2O2 的傷害有保護作用，LPS 可以誘導 NO、PGE2、
IL-6、MMP1、MMP3、MMP13 產生，但可以抑制 GAG、total collagen、TIMP-1 合成。DHEA 可以部分的減少 NO、PGE2、MMP1、MMP3、MMP13 產生，但對 IL-6 則沒有影響。所以 DHEA 在軟骨細胞受傷害的情況下具有保護作用。我們將 DHEA 的濃度提高到 10-5M，對軟骨 細胞並沒有毒性傷害，而且還可調節 MMP 與 TIMP-1 之間平衡，所以 DHEA 可扮演一個對抗 軟骨傷害的角色。
英文摘要
With increasing age, the activity of joint becomes less or pressed by gravity. For example, fatness will make cartilage atrophy, bruised and surfaces of joint will not be even, more seriously, cartilage will completely lose the function. Direct contacts among bones will lead to infection, inflammation, pains and even abnormal curves and motions. Due to aging process of body, it is impossible to generate enough proteoglycan and collagen in order to keep the structure of cartilage healthy. Thus, aging joints become less elastic, causing the bruise of joints of cartilage.
Dehydroepiandrosterone (DHEA) is an important hormone of human body which is the precursor of sexual hormone, and made from brain, skin and adrenal glands. It can also be made from Dioscorea、Taro plant or young antlers. It can be transformed into male and female hormones that are very important to the maintenance of the bone in the body. This research aims to study whether it can heal or protect chondrocytes under harmful situation or offer an alternative new treatment for cartilage damage.
Chondrocytes were isolated from mouse knee cartilage and cultured
three-dimensionally in agarose. Then simulating the proliferation of infectious arthritis and osteoarthritis by adding DHEA with LPS, or DHEA with SNAP, GAG were measured with DMB assay, total collagen with OHP assay, (NO、PGE2、IL-6、MMPs) were measured with specific kits respectively. Furthermore, the cells transcriptional expressed the detectable levels of COX-2、iNOS、MMP1、MMP3 and MMP13 mRNAs.
This experiment showed revealed that DHEA induced an increasing cell proliferation with the optimum effects at 10-6M. LPS increased the production of NO、 PGE2、
IL-6、MMP1、MMP3 and MMP13, but inhibited the synthesis of GAG、OHP and TIMP-1.
DHEA partially revered the effect of LPS on NO、PGE2、MMP1、MMP3 and MMP13 production, had no effect on IL-6 production. So DHEA has the function of protection to the newborn mice chondrocytes under the condition of being harmed. Our study demonstrated DHEA has no toxic effect on chondrocytes up to 10-5M of
concentration and has an ability to modulate the imbalance between MMPs and TIMP-1, which suggest that it has a protective role against articular cartilage loss.
