Objective To investigate the effects of hyaluronic acid against nicotine on human periodontal ligament fibroblasts,and to explore the molecular mechanism underlying nicotine-induced cytotoxicity. Methods In vitro cultured human periodontal ligament fi-broblasts(HPDLFs)were divided into four groups according to the treatment:①normal group:cultured in DMEM medium continue;②the nicotine groups:concentrations of nicotine were added respectively(10 μg/mL,100 μg/mL,250 μg/mL,500 μg/mL,1 000 μg/mL). ③hyaluronic acid groups:in DMEM hyaluronic acid concentrations were 0.1% and 0.2%;④the nicotine + hyaluronic acid groups:concentrations of 0.1% and 0.2% in DMEM HA and different concentrations of nicotine jointly developed into 10 subgroups (total). Four groups were determined by MTT method(thiazole blue tetrazoliumbromide). The cell proliferation rate,activity of ALP, the mRNA levels of Runx2,ColⅠ,OPN and OCN,and cell calcium mineralization ability were observed. Results ①Compared with the control group,the concentration of nicotine could inhibit the proliferation activity of PDLFs,alkaline phosphatase activity,and lev-els of mRNA expression of Runx2,ColⅠ,OPN and OCN. Calcification mineralized nodules were not seen in pure nicotine treatment group. ②Compared with the control group,concentrations(0.1% and 0.2%)of hyaluronic acid had an obvious promoting effect on the periodontal ligament fibroblasts,both kinds of concentrations of hyaluronic acid could promote the proliferation activity of HPDLFs and alkaline phosphatase activity,and promote the rise of Runx2,ColⅠ,the level of mRNA expression of OPN and OCN,and mineralized nodules were observed. ③Compared with the pure nicotine effect group, hyaluronic acid + nicotine group played a role in promoting HPDLFs proliferation activity and alkaline phosphatase activities, the mRNA expression level of Runx2, ColⅠ, OPN and OCN im-proved,and calcification mineralized nodules were observed. Conclusion HA can increase the activity of ALP and mineralized tissue-associated proteins,including Runx2,Col I,OPN and OCN,etc and can resist the toxic effect of nicotine on HPDLFs,thus playing a protective role.%目的 观察不同浓度的尼古丁对人牙周膜成纤维细胞(HPDLFs)增殖的影响,并探讨透明质酸对尼古丁作用下对HPDLFs的保护作用.方法 体外成功培养的HPDLFs,按不同的处理分为4组:①单纯细胞组(对照组);②尼古丁组:浓度分别为10、100、250、500、1000 μg/mL;③HA组:浓度为0.1%和0.2%;④尼古丁组+HA组(总10子组).观察4组细胞增殖率, ALP的活性,Runx2、ColⅠ、OPN、OCN的mRNA的水平,细胞钙矿化能力.结果 ①与对照组相比,各浓度尼古丁组均可以抑制HPDLFs的增殖率、ALP活性,Runx2、ColⅠ、OPN、OCN的mRNA水平,未见钙化矿化结节.②与对照组相比,HA组均可以促进HPDLFs的增殖率、ALP活性和Runx2、ColⅠ、OPN、OCN的mRNA水平并观察到了钙化矿化结节.③与尼古丁组相比, HA+尼古丁组对HPDLFs的增殖率和ALP活性起到促进作用,对Runx2、ColⅠ、OPN、OCN的mRNA的表达水平有提高并均观察到钙化矿化结节;各组差异具有统计学意义(P<0.05).结论 HA能够介导HPDLFs增殖,增加ALP活性和矿化组织相关蛋白,包括Runx2、ColⅠ、OPN和OCN等,并且抵抗尼古丁对HPDLFs的毒性作用,从而起到保护作用.
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