消炎藥物對人類成骨細胞之增殖及細胞週期調控因子之影響

The Effects of Anti-inflammatory Drugs on Proliferation and the Regulatory Factors of Cell Cycle in Human Osteoblas

李靜如(Ching-Ju Li)

指導教授 ： 何美泠

高雄醫學大學/醫學院/醫學研究所/碩士(2004年)

摘要

中文摘要
非類固醇消炎藥物(non-steroidal anti-inflammatory drugs; NSAIDs)，臨床上常用以減輕骨折或者手術後所引起之疼痛與發炎現象，其作用機制是經由抑制前列腺素合成酶－環氧化酶(cyclooxygenase)的合成，進而抑制前列腺素的生成而達到消炎止痛的作用。類固醇消炎藥物主要為糖皮質素類藥物(Glucocorticoids)，目前已廣泛的運用於化學治療及作為消炎藥物。其作用機制是經由抑制 phospholipase A2 之形成，進而抑制前列腺素之生成，達到消炎之效果。然而，已有文獻指出在活體內的研究中，類固醇及非類固醇消炎藥物均會抑制骨修復及骨生成作用。最近發展的COX-2選擇性抑制劑亦有活體內研究顯示，celecoxib與 refecoxib會抑制骨生長入移植物質之作用及骨折後的骨修復。我們之前的活體外研究中發現，非類固醇消炎藥物會抑制大鼠成骨細胞的增殖、停滯其細胞週期停於G0/G1期並且造成大鼠成骨細胞的細胞凋亡與細胞壞死。研究之最終目標乃希望能運用於人類。然而統整以上，類固醇消炎藥物、非類固醇消炎藥物及COX-2 選擇性抑制劑對於人類成骨細胞之研究至今尚未有文獻報告。本研究目的是探討類固醇消炎藥物、非類固醇消炎藥物及COX-2 選擇性抑制劑探討其對於人類成骨細胞之影響。
本研究分別以類固醇消炎藥物(dexamethasone)、四類不同結構之非類固醇消炎藥物(indomethacin、ketorolac、diclofenac及piroxicam)及COX-2 選擇性抑制劑(celecoxib及DFU)作用於人類正常成骨細胞。利用胸腺嘧啶標誌法(thymidine incorporation)，探討藥物對人類成骨細胞增殖之影響。我們運用PI (propidium iodide)將DNA染色後，再以流式細胞分析術分析人類正常成骨細胞其細胞週期之分佈百分比的改變。我們亦分析藥物處理後，人類正常成骨細胞釋放LDH (Lactate Dehydrogenase)之程度代表細胞毒性。更進一步，我們以反轉錄聚合酶鏈鎖反應(RT-PCR)探討藥物對人類正常成骨細胞的細胞週期調控因子及細胞凋亡因子基因表現之改變，以釐清其影響細胞週期停滯的可能機制。
研究結果顯示，我們所投與之四種非類固醇消炎藥物、dexamethasone及COX-2 選擇性抑制劑均顯著抑制人類成骨細胞的增殖作用。然而，當我們將celecoxib及DFU移除，再以新鮮之培養液培養24小時後發現， celecoxib處理組，其抑制人類成骨細胞增殖之作用是可以被完全回復的；但是DFU處理組中，只有10-7M及10-6 M兩組能完全回復（p< 0.01；與加藥組比較），而10-5M及10-4 M則是呈現部分回復的現象。流式細胞術的結果顯示，celecoxib、DFU、indomethacin及 dexamethasone均會造成細胞週期停滯於G0/G1期。另外，LDH釋放試驗結果顯示，在處理indomethacin、ketorolac、diclofenac及piroxicam 24小時後，並無顯著的毒殺作用。Dexamethasone也僅在較高濃度（10-6M）才會對人類成骨細胞有顯著之毒殺作用。將藥物處理時間延長至48小時後，僅有以較高濃度diclofenac（10-4M）以及dexamethasone （10-7M及10-6M）處理組有顯著之毒殺作用。然而，在給予DFU及celecoxib 24小時後，10-7M-10-4M之藥物均對人類成骨細胞有顯著之毒殺作用。在探討藥物對人類成骨細胞的細胞週期調控因子及細胞凋亡因子基因表現之影響的研究。結果顯示，dexamethasone 10-7M會顯著增加p27Kip1基因的表現，而dexamethasone 10-6M則會顯著地增加p53、p21Waf1及p27Kip1基因的表現；Indomethacin及diclofenac顯著地增加p21Waf1及p27Kip1基因的表現；celecoxib 10-5M會顯著增加p27Kip1表現；celecoxib 5x10-5M則會顯著地增加p53、p21Waf1、p27Kip1及Bax基因的表現；DFU則是顯著增加p27Kip1及Bax基因的表現。
本研究結果顯示，類固醇消炎藥物（dexamethasone）、四類不同結構之非類固醇消炎藥物（indomethacin、ketorolac、diclofenac及piroxicam）及COX-2 選擇性抑制劑（celecoxib及DFU）均會抑制人類成骨細胞之增殖，並停滯細胞週其於G0/G1期。類固醇消炎藥物及COX-2 選擇性抑制劑會對成骨細胞有顯著的毒殺作用，而非類固醇消炎藥物則無顯著之毒殺作用。類固醇消炎藥物非類固醇消炎藥物及COX-2 選擇性抑制劑可能藉由影響不同的細胞週期調控因子的表現，而抑制細胞的增殖及細胞週期的進行。根據以上本論文之結果我們推測消炎藥物對於人類成骨細胞之抑制增殖及對其造成毒殺之作用，可能會導致人體骨生成作用受到抑制。此外，類固醇消炎藥物、非類固醇消炎藥物及COX-2 選擇性抑制劑可能是透過不同的機制影響人類成骨細胞之功能。

關鍵字

細胞週期 ； 增殖 ； 人類成骨細胞 ； 消炎藥物 ； 細胞週期調控因子 ； 毒殺作用

並列摘要

Abstract
Nonsteroidal anti-inflammatory drugs (NSAIDs) block the action of cyclo-oxygenase (COX) to relieve pain and inflammation. Steroids have been reported to inhibit phospholipase A2 and reduce prostaglandin synthesis. However, it has been reported that both steroids and NSAIDs suppress bone repair and remodeling in vivo. The recent developed selective COX-2 inhibitors, such as celecoxib and refecoxib were also reported to decrease bone repair in animal studies. Our previous studies showed that NSAIDs inhibited osteoblast proliferation and induced cell death in fetal rat osteoblast cultures. However, the effects of steroid, NSAIDs and COX-2 inhibitors on the proliferation and cell death of human osteoblast are rarely investigated.
In this study, the effects of steroid, NSAIDs and COX-2 selective inhibitors on the functions of human osteoblasts are examined. Normal human osteoblasts were purchased from Clonetics®. Indomethacin, ketorolac, piroxicam and diclofenac (10-5 and 10-4M); dexamethasone (10-7 and 10-6M); Celecoxib and DFU, an analogue of refecoxib, (10-7-10-4M) were used in this study. Drugs were treated for 24 or 48 hr in human osteoblast cultures. DNA synthesis was examined by thymidine incorporation. Cell cycle kinetics was measured by flow cytometry. Cytotoxicity was tested by lactate dehydrogenase (LDH) leakage. RNA expressions of cell cycle regulators and pro-apoptotic factors were analyzed by RT-PCR.
Our results showed that the tested 4 NSAIDs, dexamethasone and both the COX-2 selective inhibitors significantly inhibited proliferation of human osteoblast. The results of cell cycle kinetics showed that the tested 4 NSAIDs, dexamethasone and COX-2 selective inhibitors arrested cell cycle of human osteoblasts at G0/G1 phase. The inhibition effect on proliferation of celecoxib was completely reversed at 24 hr after removal of the agents, while DFU was partially reversed.
Our results showed that celecoxib and DFU significantly increased LDH leakage in cultures upon 24 hr treatments, while NSAIDs and dexamethasone showed non-significant effects. After 48 hr treatments, 10-4M of diclofenac and 10-7-10-6 M of dexamethasone showed cytotoxic in human osteoblast cultures. Furthermore, our results showed that dexamethasone 10-7M increased the mRNA expression of p27Kip1 (Cdk inhibitors). Dexamethasone 10-6M increased the mRNA expressions of p53, p21Waf1 and p27Kip1. Indomethacin and diclofenac significantly increased the mRNA expressions of p21Waf1 and p27Kip1. Celecoxib 10-5M increased the mRNA expression of p27Kip1, while 5x10-5M of celecoxib increased the mRNA expressions of p53, p27Kip1, p21Waf1 and Bax. However, DFU only significantly increased the mRNA expressions of p27Kip1 and Bax.
In this study, we found that NSAIDs (indomethacin, ketorolac, diclofenac and piroxicam), steroid (dexamethasone) and COX-2 inhibitors (celecoxib and DFU) all significantly inhibited proliferation and arrested cell cycle at G0/G1 phase. Steroid and COX-2 inhibitors had cytotoxicity in human osteoblasts. However, NSAIDs had no cytixic effects in human osteoblasts. The effects of NSAIDs, steroid and COX-2 inhibitors on proliferation and cell cycle progression in human osteoblasts may undergo through went influencing the expressions of different cell cycle regulators and pro-apoptosis factors. The results suggest that the effects of anti-inflammatory drugs on the proliferation inhibition and cytotoxicity of human osteoblasts may cause the suppression of bone formation. These effects of steroid, NSAIDs and COX-2 inhibitors on osteoblastic functions may through different mechanisms.

並列關鍵字

cell cycle regulators ； cytotoxicity ； Anti-inflammatory drugs ； human osteoblasts ； cell cycle ； proliferation

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