於機械力或機械力合併細菌內毒素的刺激下RAW264.7 Cells 之核因子活化因子受體及COX-2 訊息核糖核酸表現的探討

研究目的：於機械力或機械力合併細菌內毒素(lipopolysaccharide；LPS)的刺激下RAW264.7 cells 之核因子活化因子受體(RANK)及COX-2 訊息核糖核酸(mRNA)表現的探討。材料與方法：本實驗使用RAW264.7 cells(蝕骨細胞之前驅細胞)進行培養，先以LPS 0.1μg/ml、1μg/ml (取自E.coli)或張力2%，頻率1Hz；張力18%，頻率0.1Hz分別對RAW264.7 cells進行不同時間點的刺激，觀察RANK 及COX-2 mRNA的表現。之後再合併LPS和張力一起刺激RAW264.7 cells，研究RANK 及COX-2 mRNA的表現。接著使用RT-PCR闡明RANK 及COX-2 mRNA在接受不同刺激後的表現情形，基因表現相對量則以比較性Ct 法分析(Comparative Ct method)計算之。最後以JMP軟體進行統計分析。結果：單獨LPS刺激的實驗中，都無法得到RAW264.7 cells 的RANK/ COX-2 mRNA的明顯表現差異。但是，單獨張力刺激下，不管是以張力2%，頻率1Hz 或是張力18%，頻率0.1Hz的條件刺激RAW264.7 cells，都可以觀察到在24小時間歇性的刺激後，RANK mRNA的顯著表現差異(p <0.05)。而且RANK mRNA的表現和時間點呈現顯著的正相關性。但是，不管是以張力2%，頻率1Hz 或是張力18%，頻率0.1Hz的條件刺激RAW264.7 cells，都無法在2，12，24小時，這三個時間點，觀察到COX-2 mRNA的顯著表現差異(p >0.05)。LPS合併張力刺激下，RANK mRNA濃度的表現確實和單獨刺激下有所不同：三個時間點2，6，24小時的Ct值和基準值比起來並無顯著差異(p >0.05)。不過在12小時的間歇性刺激後，得到RANK mRNA的Ct值都有小於基準值的傾向。RANK mRNA的表現和這三個時間點也有負相關性。不過，和單獨刺激相同的，仍然無法觀察到COX-2 mRNA表現差異性。結論： LPS單獨的刺激下，都沒有辦法觀察到RANK和COX-2 mRNA的顯著表現差異。因此推論以LPS刺激單獨培養的RAW264.7 cells，無法激發RANK/RANKL此一路徑。蝕骨前驅細胞在此路徑並非起始者，可能需要成骨細胞傳達訊息給蝕骨前驅細胞才可以進一步引發骨吸收。不過，單獨培養的RAW264.7 cells，在張力的刺激下，是不需經由成骨細胞先提供訊息。由本實驗可知於機械力的刺激下，蝕骨前驅細胞可獨立運作，表現RANK mRNA而且此路徑和COX-2無直接相關。雖然在人體內成骨細胞和蝕骨前驅細胞是共同存在的，不過單獨的培養和刺激下可以幫助釐清這兩種細胞在不同刺激下扮演的角色。然而最後LPS合併張力的實驗下，RANK mRNA的表現在早期尤其是第12小時有被抑制的情形，根據LPS和張力單獨實驗的結果，推論是LPS在早期抑制了RANK mRNA的表現。雖然本研究都無法在RAW264.7 cells上，找出COX-2和RANK的相關性，不過從實驗結果得知張力可以直接啟動RANK mRNA的表現，而LPS在早期會抑制RANK mRNA的表現。

關鍵字

機械力；細菌內毒素；蝕骨前驅細胞

並列摘要

Objective: The Expression of the RANK/COX-2 mRNA by mechancial force alone or combination with LPS in RAW264.7 Cells Material and Methods: The RAW264.7 cells (osteoclast precursor cells) were used in the current investigation. First, we observe the RANK and COX-2 mRNA expression by LPS 0.1μg/ml, 1μg/ml (from E.coli) or the tension 2 % (1Hz), 18% (0.1Hz) for different time stimulation. Then merge again LPS 0.1μg/ml and the tension to stimulate RAW264.7 cells together to study the RANK and COX-2 mRNA expression. RT-PCR procedure was used to quantify the RANK and COX-2 mRNA expression after different stimulation. The relative quantity of gene expression was calculated by Comparative Ct method and was analyzed by JMP statistical software. Results: In RAW264.7 cells, we were unable to discover any obvious changes of the RANK or COX-2 mRNA expression by LPS alone. But, there were obvious expression of the RANK mRNA in RAW264.7 cells by tension of 2%, 1Hz and 18%, 0.1Hz alone after 24 hours intermittent stimulation(p <0.05). Moreover the expression of the RANK mRNA and the irritation periods had significant positive relevance. However, there was no significant expression of the COX-2 mRNA by tension alone (p >0.05). Furthermore the expression of the RANK mRNA was truly different between combination and single stimulation with LPS or tension force. In the combination stimulation of 2, 6 and 24hours, the Ct values were the same as datum value (p >0.05). Nevertheless the Ct values had a trend of smaller than the datum value after 12 hours intermittent stimulation and the expression of the RANK mRNA and the irritation periods (2, 6, 12 hours) present some negative correlation. There was still no obvious expression of the COX-2 mRNA. Conclusion: There was no obvious expression of the RANK or COX-2 mRNA, so LPS alone may not evoke the pathway of RANK/RANKL in RAW264.7 cells. Osteoclast precursor cells are not the initiator and they may need osteoblasts to give information to them for initiating bone resorption by LPS. However, there was obvious expression of the RANK mRNA in RAW264.7 cells by tension without the presence of osteoblasts. By tension alone, RAW264.7 cells could release the RANK mRNA independently and this pathway is unrelated with COX-2. Although the osteoblasts and the osteoclast precursor cells coexist in the human body, it may help us to define the roles of these cells play in different stimulation. At last, in the combination stimulation of LPS and tension, the RANK mRNA was suppressed in early stage, in particularly the 12th hour. From the data of the present study, we speculated that LPS has the suppression effect on the RANK mRNA in early stage. Although in RAW264.7 cells we were unable to observe the relevance of the COX-2 and RANK mRNA, we found that tension can initiat the activity of the RANK mRNA and LPS may suppress the expression of the RANK mRNA in the early experiment periods.