淫羊藿苷对人成骨细胞增殖及OPG蛋白表达的实验研究

Experimental study on the mechanism of icariin improving human osteoblasts proliferation and the expression of OPG protein

目的:建立体外培养人成骨细胞系,观察淫羊藿苷对人成骨细胞增殖的影响,以及对人成骨细胞内骨保护素(Osteoprorotegerin,OPG)蛋白表达的影响,探讨淫羊藿苷促进人成骨细胞骨形成的可能作用机制。方法:将手术中取得的人股骨头松质骨骨片,使用酶消化法进行培养,取生长良好的第3代人成骨细胞进行实验。实验分为4组,对照组给予15%NCS-DMEM-F12(1:1)培养液培养;淫羊藿苷组分别于正常培养液内添加终浓度为10-6、10-8、10-10mol/L淫羊藿苷。噻唑蓝比色法(MTT)分别在培养1、3、5、7、9d后观察各组人成骨细胞的增殖情况,用蛋白免疫印迹方法(in-cell western blot)测定各组培养8、10、12d后人成骨细胞内OPG蛋白的表达情况。采用重复测量的方差分析方法,比较各组间在不同时间点的作用差异。结果:MTT结果,淫羊藿苷组具有促进人成骨细胞增殖的作用,并呈明显的量效关系,即随着淫羊藿苷浓度的增高,其促人成骨细胞增殖的能力增强,以10-6mol/L淫羊藿苷组(0.402±0.033)促成骨细胞增殖的作用最显著,与对照组(0.268±0.031)比较差异有统计学意义(P<0.05)。在时效关系的研究中,随着培养天数的延长,人成骨细胞的量逐渐增加,人成骨细胞于第5天时进入快速生长期,第7天进入平台期;淫羊藿苷组从第5天开始促进人成骨细胞的增殖,第7、9天仍然具有明显的促成骨细胞增殖的作用,以第9天促增殖作用最强。其中,第9天10-6mol/L淫羊藿苷组(0.402±0.033)与对照组(0.268±0.031)比较有统计学意义(P<0.01)。OPG表达结果,对照组人成骨细胞培养至第8天时检测到OPG蛋白的表达,表达量为1.01±0.08,第12天达到表达高峰为1.80±0.10,两个值比较有统计学差异(P<0.05);在观察的不同天数内,不同浓度的淫羊藿苷组人成骨细胞内OPG蛋白表达低于对照组,且随着淫羊藿苷浓度的降低,OPG蛋白的表达量逐渐降低,差异有统计学意义(P<0.05);干预12d,10-6mol/L淫羊藿苷组OPG量(1.67±0.13)和10-8mol/L淫羊藿苷组OPG量(1.64±0.05)比较,无统计学差异(P>0.05)。结论:淫羊藿苷促人成骨细胞骨形成能力的作用途径之一,是通过提高人成骨细胞的增殖能力而实现。

Objective：To establish the human osteoblasts culture system in vitro,observe the effects of icariin on human osteoblasts proliferation and expression of OPG protein,and to explore the mechanism of promoting bone formation about human osteoblast in icariin.Methods：The femoral cancellous bone pieces were obtained from the operation.The enzyme digestion method was used for culturing.The third passage of human osteoblast was taken for experiments.The cells were divided into four groups,the control group was treated with 15%NCS-DMEM-F12（1：1）,the experimental groups were respectivelytreated with 10-6,10-8,10-10 mol/L icariin.The MTT method was used to observe the proliferation of human osteoblast on 1,3,5,7,9 d;in 8,10,12 d,western blot was used to determine the expression of OPG protein on human osteoblast.Results：①Results of MTT：the icariin promoted the proliferation of human osteoblast.There was a concentration-response relation,while with the concentration of icariin increased,the ability was more obvious.There was statistically difference between 10-6 mol/L icariin group（0.402±0.033） and the control group（0.268±0.031）（P〈0.05）.In the timely research,as the time prolong,the number of human osteoblast were more.At the fifth day,the human osteoblast entered rapid growth period,and access the growth platform stage;the icariin began to promote the proliferation of human osteoblast from the fifth day,which almost maintained to the 7th day and the 9th day,and most obvious in the 9th day.There was statistically difference between 10-6mol/L icariin group（0.402±0.033） and the control group（0.268±0.031） at the 9th day.②The results of OPG protein expression：in the control group,the expression of OPG protein was detected at the 8th day（1.01±0.08）,and reached the expression peak（1.80±0.10）,there was statistically different（P〈0.05）.In the different days and different concentration icariin groups,the expressions of OPG protein were all inferior to the control group.While the concentration decreasing,the expression was less.There were statistically difference（P〈0.05）.At the day 12,there was no significant difference of OPG protein expression between the 10-6mol/L icariin group and the 10-8mol/L icariin group（P〈0.05）.Conclusion：The effect of icariin promoting the proliferation of human osteoblast maybe is one of the mechanisms of improving the bone formation on human osteoblast.