消炎藥物對人類關節軟骨細胞之正常功能及終止分化之標的基因表現之影響

Effects of anti-inflammatory drugs on the expressions of normal functional genes and marker genes of terminal differentiation in cultured human articular chondrocytes

張玲華(Ling-Hua Chang)

指導教授 ： 何美泠

高雄醫學大學/醫學院/醫學研究所/碩士(2005年)

摘要

類固醇和非類固醇消炎藥物(non-steroidal anti-inflammatory drugs; NSAIDs)，臨床上常用於治療退化性關節炎所引起的疼痛與發炎現象。然而，在過去活體內的研究文獻中報告，類固醇會抑制軟骨細胞的增殖及降低glycosaminoglycan的合成速率。非類固醇消炎藥物也會抑制軟骨細胞間質的合成。近年來由於COX-2選擇性抑制劑對胃腸有較少的副作用，已經成為治療退化性關節炎的新處方。然而，COX-2選擇性抑制劑在正常的人類關節軟骨細胞方面的影響較少被研究。另一方面，過去的研究發現，導致退化性關節炎的病理機制之一，是關節軟骨細胞進行類似於軟骨內骨化時之終止分化，造成細胞肥大，礦質沈積，最後導致細胞死亡。因此本研究欲探討消炎藥物於減輕退化性關節炎的發炎與疼痛的同時，藥物是否影響軟骨細胞之正常功能及終止分化時之標的基因的表現。
本研究採用分離自正常人類關節之軟骨細胞（23歲男性腺體之膝部軟骨），以活體外立體培養的細胞培養模式，研究探討三類的消炎藥物，分別為類固醇：dexamethasone (10-9~10-7M)；四種常用的NSAIDs: diclofenac, indomethacin, ketorolac, piroxicam（10-5~10-4M）以及兩種COX-2選擇性抑制劑：celecoxib 和DFU (rofecoxib的analogue) (10-6~10-5M)，連續作用1, 5, 7天。本研究所測定的正常的關節軟骨細胞之功能基因包括SOX9、第二型膠原蛋白和蛋白多醣（aggrecan）；而終止分化時之標的基因為第X型膠原蛋白、PTHrP、Ihh。以反轉錄聚合酶鏈鎖反應(RT-PCR)測定功能基因及終止分化時的標的基因之mRNA表現是否有受到藥物作用的影響。以DMMB（dimethylmethylene Blue）分析法測定間質中所含的硫化醣蛋白之總量。並且探討消炎藥物對關節軟骨細胞之細胞毒性之影響，以乳酸去氫酵素（lactate dehydrogenase）漏出量代表細胞毒性。以TUNEL 染色觀察細胞凋亡。
本研究結果顯示，在連續處理dexamethasone (10-9~10-7M), DFU and celecoxib (10-6~10-5M) 1, 5, 7天後，均顯著地抑制SOX9, type II collagen和aggrecan mRNA的表現。然而非選擇性NSAIDs, diclofenac, indomethacin, ketorolac, piroxicam（10-5~10-4M）對type II collagen, SOX9和aggrecan mRNA的表現之作用，是藥物處理1, 5天後有抑制作用，但處理第7天則無顯著的影響。在測定硫化醣蛋白的總量之結果，也顯示dexamethasone (10-9~10-7M), DFU and celecoxib (10-6~10-5M)會抑制軟骨細胞的硫化醣蛋白之含量，而處理diclofenac, indomethacin, ketorolac, piroxicam （10-5~10-4M）後1天或 5天均有抑制作用，但處理7天則無顯著的影響。此結果與基因表現的結果是一致性的。更重要的是，我們發現處理這三類消炎藥物都促進type X collagen和抑制PTHrP mRNA的表現。三類消炎藥物在細胞毒殺與細胞凋亡方面之作用，除了celecoxib在10-4M有顯著的細胞毒殺作用之外，其他均無顯著的毒殺或引導關節軟骨細胞之凋亡。綜合以上結果，我們推論類固醇及COX-2選擇性抑制劑會抑制細胞外間質的合成，推測藥物可能是透過抑制SOX9這個transcription factor (轉錄因子)的表現而抑制type II collagen及aggrecan的表現。此外，本研究所使用之三類消炎藥物都會促進終止分化時的基因表現，推論消炎藥物可能有助於關節軟骨細胞走向終止分化。

關鍵字

終止分化 ； 消炎藥物 ； 人類關節軟骨

並列摘要

Steroidal and nonsteroidal anti-inflammatory drugs (NSAIDs) block the action of cyclo-oxygenase (COX) to relieve pain and inflammation for osteoarthritis (OA) patients. However, investigators have indicated that NSAIDs might damage the cartilage. Previous in vivo studies demonstrated that several glucocorticoids reduce proliferation of chondrocytes and the synthetic rate of glycosaminoglycan in articular cartilage. Previous in vitro studies also demonstrated that several NSAIDs accelerate the damage of osteoarthritic cartilage by ways of inhibiting the synthesis of proteoglycan and/or facilitating the effects of cytokines. Recently, COX-2 inhibitors have been prescribed for long-term treatment of OA for their lesser side effects on gastrointestines. However, the influences of COX-2 inhibitors on normal articular chondrocytes were rarely investigated. On the other hand, it has been found that one of the main pathogenesis of OA is that articular chondrocytes undergo terminate differentiation, so that the chondrocytes become hypertrophy, synthesize collagen type X, form mineral formation and eventually undergo apoptosis. However, the influences of anti-inflammatory drugs on this terminal differentiation of articular chondrocytes remain unclear. Accordingly, in this study, we investigated the effects of anti-inflammatory drugs on the expressions of normal functional genes and the marker genes of terminal differentiation in cultured human articular chondrocytes. Cytotoxicity and apoptosis induced by anti-inflammatory drugs on chondrocytes were also tested.
In this study, we isolated human articular cartilage from a 23-year-old male cavader and encapsulated chondrocytes in alginate beads as a 3 dimensional culture. The effects of dexamethasone (10-9~10-7M); diclofenac, indomethacin, ketorolac and piroxicam（10-5~10-4M）; and DFU (an analogue of refecoxib) and celecoxib (10-6~10-5M) were tested. The expressions of normal functional genes, collagen type II, SOX9, aggrecan, and the marker genes of terminal differentiation, collagen type X, Indian hedgehog (Ihh) and PTHrP, were examined in human articular chondrocytes by RT-PCR. Total sulfated glucosaminoglycan was quantified by DMMB assay. Cytotoxicity was tested by lactate dehydrogenase (LDH) leakage. Apoptosis was examined by TUNEL stain.
Our results showed that 1-, 5- and 7-day treatments of dexamethasone (10-9~10-7M), DFU and celecoxib (10-6~10-5M) significantly inhibited the expressions of collagen type II, SOX9 and aggrecan. However, non-selective NSAIDs, diclofenac, indomethacin, ketorolac and piroxicam （10-5~10-4M）, showed no significant effect on the expression of these functional genes of articular chondrocytes. We also found that the three classes of anti-inflammatory drugs induced the mRNA expressions of collagen type X, but inhibited that of PTHrP. The result of DMMB assay showed that dexamethasone (10-9~10-7M), DFU and celecoxib (10-6~10-5M) significantly inhibited the total sulfated glucosaminoglycan content. However, the treatment of either diclofenac, indomethacin, ketorolac or piroxicam （10-5~10-4M）for 7 days showed no significant effect in comparison to the control cultures. The results of glycosaminoglycan were consistent with the gene expression of aggrecan. Our results showed that anti-inflammatory drugs showed no significantly effects on cytotoxicity and apoptosis of chondrocytes.
These results showed that dexamethasone and COX-2 inhibitors inhibited functional genes expressions, suggesting that dexamethasone and COX-2 inhibitors may inhibit the synthesis of extracellular matrix in normal human articular chondrocytes. We also suggest that these drugs may inhibited the gene expressions of type II collagen and aggrecan through inhibiting SOX9 expression. In addition, these three classes of anti-inflammatory drugs induced marker genes of terminal differentiation, suggesting that they may help to promote cell undergoing terminal differentiation.

並列關鍵字

anti-inflammatory drugs ； Human articular chondrocyte ； terminal differentiation

參考文獻

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