【作者】 孙海博； 佟美辰； 刘世杰； 牛卫东； 王丽娜；

【Author】 Sun Haibo;Tong Meichen;Liu Shijie;Niu Weidong;Wang Lina;School of Stomatology, Dalian Medical University;

【机构】 大连医科大学口腔医学院；

【摘要】 目的:探究苗药固齿方药煎液对于成骨细胞与破骨细胞增殖、分化的影响,为将中药复方制剂用于临床上治疗口腔骨破坏疾病提供实验依据。方法:(1)苗药固齿方药煎液的制备:取青盐15g、生石膏15g、续断12g、散血飞5g、对叶莲5g、骚羊古8g、艾纳香8g、卷柏8g、鸡屎藤5g.加十倍量的蒸馏水浸泡两小时后大火煮沸,煎煮30分钟两次。冷却至室温后加入95%乙醇沉淀,离心后取上清,煎煮浓缩至1g/ml作为原液。经微孔滤膜过滤细菌,装入无菌瓶备用。(2)细胞增殖实验:小鼠成骨前体细胞系MC3T3-E1、小鼠破骨前体细胞系RAW264.7复苏传代后,铺96孔板,设置7个药煎液浓度梯度,分别为0、1×10-1、1×10-2、1×10-3、1×10-4、1×10-5、1×10-6,于0h、24h、48h、72h测定细胞增殖情况。(3)细胞分化实验:MC3T3-E1细胞、RAW264.7细胞复苏传代后铺于六孔板中,铺板后7天后MC3T3-E1细胞进行ALP染色、RAW264.7细胞进行TRAP染色,观察其向成骨细胞与破骨细胞的分化情况。结果:MC3TCE1细胞在药物浓度为1×10-4时,细胞于48h、72h时增殖明显(p<0.05)。RAW264.7细胞,药物浓度大于或等于1×10-2可明显抑制细胞增殖(p<0.05),药物浓度小于1×10-4时,对细胞增殖无影响(P>0.05)。ALP染色结果显示实验组ALP的表达明显高于空白对照组(p<0.05)。TRAP染色结果显示实验组与对照组阳性多核细胞数量无统计学差异(P>0.05)。结论:苗药固齿方能够促进MC3T3-E1细胞增殖与分化,同时可抑制RAW264.7细胞的增殖,对RAW264.7的分化无明显影响。苗药固齿方可能具有成骨作用。更多还原

【Abstract】 Objective:To investigate the effect of decoction of Miao medicine Guchi formula on proliferation and differentiation of osteoblasts and osteoclasts,and to provide experimental basis for the clinical treatment of oral bone destruction diseases with Chinese herbal compound preparation.Methods:(1) preparation of decoction of Miao medicine Guchi formula:15 g of green salt,15 g of raw gypsum,12 g of rhizoma xanthii,5 g of sanxue fei,5 g of Lythrum salicaria L,8 g of saoyang gu,8 g of Blumea balsamifera,8 g of selaginella and 5 g of jishiteng.Soak in 10 times the amount of distilled water for two hours,then bring to a boil on high fire and fry for 30 minutes twice.After cooling to room temperature,95% ethanol was added to precipitate,supernatant was taken after centrifugation,and the supernatant was boiled and concentrated to 1 g/mL as the original liquid.Bacteria were filtered by microporous membrane and put into sterile bottles for use.(2) Cell proliferation test:After mouse osteogenic precursor cell line Mc3 t3-el and mouse osteoclast precursor cell line RAW264.7 were cultured,96-well plates were laid and 7 concentration gradient of decoctions were set,which were 0,1×10-1,1×10-2,1×10-3,1×10-4,1×10-5,1×10-6,respectively.Cell proliferation was measured at 0 h,24 h,48 h and 72 h.(3) Cell differentiation experiment:Mc3 t3-el cells and RAW264.7 cells were revived and cultured and placed in a six-well plate.ALP staining was performed on Mc3 t3-el cells and TRAP staining was performed on RAW264.7 cells 7 days after plate placement to observe the differentiation of Mc3 t3-el cells into osteoblasts and osteoclasts.Results:Mc3 tc-el cells proliferate significantly at 48 h and 72 h when drug concentration was1×10-4(p <0.05).The proliferation of RAW264.7 cells was significantly inhibited when the concentration of RAW264.7 was greater than or equal to 1×10-2(P <0.05),but had no effect when the concentration was less than 1×10-4(P> 0.05).ALP staining results showed that the expression of ALP in experimental group was significantly higher than that in blank control group(P <0.05).TRAP staining results showed that there was no statistical difference in the number of positive multinucleated cells between the experimental group and the control group(P> 0.05).Conclusion:Miao medicine Guchi formula can promote the proliferation and differentiation of Mc3 t3-e1 cells,and inhibit the proliferation of RAW264.7 cells,but has no significant effect on the differentiation of RAW264.7 cells.Miao medicine Guchi formula may have osteogenic effect.更多还原