- 學位論文
非類固醇消炎藥物對胎鼠骨骺-關節軟骨細胞之毒殺作用及細胞死亡之影響
Effects of Non-steroidal Anti-inflammatory Drugs on Cytotoxicity and Cell Death in Cultured Fetal Rat Epiphyseal-Articular Chondrocytes
摘要
中文摘要: 非類固醇消炎藥物(Nonsteroidal anti-inflammatory drugs; NSAIDs)，常使用於骨損傷的病人，以減輕骨折或手術後所引起的病痛以及發炎現象。然而，許多研究報告指出，有些NSAIDs有抑制骨生長和骨修復(bone repair)與正常骨之骨重塑(bone remodeling)的現象。COX-2選擇性抑制劑也被認為有抑制骨修復的作用。我們實驗室過去的動物活體內研究報告也顯示，NSAIDs中的ketorolac有抑制骨修復的作用，並且發現其影響骨修復的主要時機是在於骨修復早期，延遲了軟骨內骨化 (endochondral ossification)之過程。我們在過去的活體外研究發現，ketorolac和indomethacin這兩種NSAIDs會抑制成骨細胞(osteoblast)之增殖作用，同時也造成成骨細胞細胞凋亡(apoptosis)與壞死(necrosis)。然而在骨修復的早期，軟骨細胞(chondrocyte)亦扮演一個重要的角色，我們實驗室過去的研究中也發現indomethacin和ketorolac抑制軟骨細胞(chondrocyte)之增殖作用，並使得軟骨細胞之細胞週期停留在G0/G1時期。因此本研究進一步假設NSAIDs亦可能引導軟骨細胞之死亡。 本論文主要探討NSAIDs是否會毒殺軟骨細胞死亡，並進一步探討其細胞死亡之型式是凋亡或壞死。軟骨細胞之來源是取自懷孕21天胎鼠(Sprague-Dawley rats)分化中的骨骺-關節軟骨(epiphyseal-articular cartilage)，給予四種常用之NSAIDs (10-6~10-4 M): indomethacin, ketorolac, diclofenac, piroxicam 及兩種COX-2選擇性抑制劑(10-6~10-4M): celecoxib and DFU [5,5-dimethyl-3-(3-flurophyenyl)-4-(4-methylsulphonyl)phenyl-2(5H)- furanone], DFU是 rofecoxib之analogue 作用17或24小時後與未加藥組作對照比較，觀察NSAIDs對軟骨細胞之細胞毒性再進一步探討細胞死亡的形式。測定乳酸去氫酵素(lactate dehydrogenase)漏出量代表細胞毒性。以TUNEL 染色(terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling)及Caspase 3 activity觀察細胞凋亡。PI(propidium iodide)與Annexin V-FITC 雙重染色測試細胞死亡及死亡型式。 本研究之結果顯示，indomethacin， diclofenac， ketorolac， piroxicam(10-6-10-4M)皆對軟骨細胞造成細胞毒性。而celecoxib 10-5-10-4M 有細胞毒性，然10-6M之治療劑量下celecoxib和DFU則無細胞毒性。TUNEL染色結果，顯示四種NSAIDs皆能引導軟骨細胞凋亡。Caspase 3 activity也顯示四種NSAIDs造成軟骨細胞凋亡。PI與Annexin V-FITC雙重染色後之流式細胞儀之分析結果顯示四種NSAIDs: indomethacin, diclofenac, ketorolac, piroxicam皆有引導軟骨細胞凋亡及壞死的現象。根據以上本論文之結果我們推論，NSAIDs對軟骨細胞之引導死亡之作用，可能是在活體中延遲軟骨內骨化重要的原因之一。COX-2抑制劑對骨修復的影響可能不牽涉軟骨細胞之死亡。
並列摘要
ABSTRACT Nonsteroidal anti-inflammatory drugs have been widely prescribed for orthopaedic patients to relieve pain and inflammation post-operatively. However, it has been demonstrated that NSAIDs suppress bone repair and remodeling in vivo. Other reports also showed that COX-2 selective inhibitors inhibited fracture healing in vivo. We have reported that ketorolac, one of the most useful NSAIDs prescribed post-operatively, inhibits bone repair in vivo and its main effective timing is at the early stage of endochondral bone formation during the bone repair process. Furthermore, our previous results showed that ketorolac and indomethacin inhibited osteoblast proliferation and induce cell death (apoptosis and necrosis) in vitro. Chondrocytes also play an important role during bone repair, especially at early stage of repair process. Our previous study found that indomethacin and ketorolac inhibited chondrocyte proliferation and arrested cell cycle in G0/G1 phase. In this study, we further hypothesized another possible mechanism of these NSAIDs might induce chondrocytic death. In this thesis, we investigated the cytotoxic effects of NSAIDs on chondrocytes. Non-selective NSAIDs; indomethacin, ketorolac, diclofenac, and piroxicam, COX-2 selective inhibitors were celecoxib and DFU[5,5-dimethyl-3-(3-flurophyenyl)-4-(4-methylsulphonyl)phenyl -2 (5H)-furanone], an analogue of rofecoxib. Epiphyseal-articular chondrocytes were obtained from the knee of fetal rats. Cells were cultured in Dulbeco’s Modified Eagle’s Medium (DMEM) with 10% of fetal calf serum. Cultures were treated with indomethacin, ketorolac, diclofenac, piroxicam, celecoxib or DFU (10-6-10-4M) for 17 or 24 hr. Lactate dehydrogenase (LDH) leakage was tested for the cytotoxicity of NSAIDs. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase 3 activity was used for staining the apoptotic cells in cultures. Flow cytometry (Annexin V-FITC/ propidium iodide assay) was used to determine the ratio of apoptotic and necrotic cells. The results showed that all the 4 tested NSAIDs (10-6-10-4M) had significant cytotoxic effects on epiphyseal-articular chondrocytes at concentration range covering the theoretic therapeutic concentration (10-5M). However, DFU (10-6M) and celecoxib (10-6M) showed no cytotoxic effect on chondrocytes at theoretic therapeutic concentration (10-6M). We also found that 4 tested NSAIDs induced chondrocytic apoptosis and necrosis. These results suggest that the effects on cell death induction of NSAIDs on chondrocytes may play an important role on their suppressive effects during endochondral ossification of bone repair. The suppressive effects of COX-2 selective inhibitors on bone repair may not through the induction of chondrocytic cell death.