塞来昔布对大鼠骨髓间充质干细胞增殖、成骨分化的影响

Effect of Celecoxib on the Proliferation,Psteogenic Differentiation of BMSCs

目的观察不同浓度的塞来昔布对rBMSCs的增殖、成骨诱导分化的影响,探讨塞来昔布影响rBMSCs成骨诱导分化的可能机制。方法采用全骨髓贴壁法获得rBMSCs;使用不同浓度的塞来昔布(0、12.5、25、50、100μmol/L)干预培养rBMSCs,检测不同浓度的塞来昔布对rBMSCs增殖的影响;采用较高浓度的塞来昔布(100μmol/l)诱导rBMSCs成骨分化,进行ALP、茜素红染色,应用real-time PCR扩增ALP、OCN。结果细胞增殖抑制实验表明100μmol/L塞来昔布对rBMSCs增殖抑制明显(P<0.05);12.5、25以及50μmol/L塞来昔布对rBMSCs增殖抑制无影响。ALP染色结果表明,成骨诱导3天对照组以及实验组ALP染色均为阴性;诱导7天对照组ALP染色为阳性,而实验组染色为阴性;诱导14天对照组ALP染色为阳性,实验组染色阴性。茜素红染色结果表明,成骨诱导3天、7天对照组以及实验组染色均为阴性;诱导至14天对照组染色阳性,实验组染色呈阴性。荧光定量PCR结果表明,成骨诱导3天对照组以及实验组ALP mRNA、OCN mRNA表达均较低;成骨诱导7天对照组ALP mRNA表达明显升高,实验组ALP mRNA表达升高趋势较弱,对照组OCN mRNA表达与第3天相比基本无差异,实验组OCN mRNA表达下降明显;诱导14天对照组ALP mRNA表达出现明显下降,实验组ALP mRNA表达与第7天相比无明显差异,对照组OCN mRNA表达进一步升高,实验组OCN mRNA表达与第7天相比基本无变化。结论本实验发现,高浓度的塞来昔布(100μmol/L)对rBMSCs增殖具有显著抑制效应。高浓度的塞来昔布(100μmol/L)能够抑制rBMSCs成骨诱导分化过程。高浓度的塞来昔布(100μmol/L)能够抑制rBMSCs成骨诱导分化过程中ALP以及OCN mRNA表达,表明较高浓度塞来昔布抑制rBMSCs成骨诱导分化的效应可能通过抑制ALP以及OCN的基因表达实现,提示在应用NSAIDs时应注意剂量等问题,尤其是应避免使用高剂量甚至是超高生理剂量进行药物镇痛治疗。

Objective The purpose of this study was to explore the effects of different concentrations of eelecoxib on the proliferation and differentiation of rBMSCs. Methods The cells were separated through whole bone marrow adherence method. The cells were cultured with different concentrations of celeeoxib for 24 and 48 hours and then the cell proliferation was determined by MTS. The rBMSCs were cultured in the osteogenic solution with or without of celecoxib and then the ALP and alizarin red staining were performaned at 3,7,14 clays respectively. The total RNA were extracted at 3,7,14 days respectively and then to amplificate the ALP, OCN, and β -actin with Real- time PCR. Results The cell proliferation inhibition experiment showed that the proliferation of rBMSCs were significantly inhibited by 100μmol/L eeleeoxib. The ALP and Alizarin Red staining staining results showed that the activity of ALP expression and calcium deposition of rBMSCs were significantly inhibited by the higher doses of celecoxib. Further quantitative PCR showed that the mRNA expression of ALP and OCN on rBMSCs were significantly inhibited by the higher doses of celecoxib. Conclusion The cell proliferation inhibition experiment showed that the inhibitory effect on cells was more obvious in 100μmol/L of celecoxib and according to wich we showed be cautious of dosage and duration of drug using during fractures analgesic treatment. The ALP and Alizarin Red staining results showed that higher doses of celecoxib may affected the process of osteogenic differentiation on rBMSCs. The quantitative PCR showed that the higher doses of celecoxib may affected the process of osteogenic differentiation through inhibiting the mRNA expression of ALP and OCN.