益骨胶囊含药血清对成骨细胞骨保护蛋白mRNA表达的影响

Effect of Yigu capsule drug-containing serum on the expression of osteoprotegerin mRNA in osteoblasts

目的:观察益骨胶囊含药血清对体外培养的大鼠成骨细胞骨保护蛋白mRNA的影响。方法:实验于2004-05/2005-02在广州暨南大学生命科学技术学院细胞与蛋白质工程实验室完成。选取12月龄SD雌性大鼠20只,随机分为2组。中药给药组(益骨胶囊由淫羊藿、当归、白芍、枸杞子等8味中药组成)和蒸馏水对照组,每组10只。运用大鼠灌胃的方法制备含药血清和对照血清,分离、培养新生大鼠的颅骨成骨细胞,传代后分为2组(含药血清组和空白血清对照组),每组8孔,分别在培养24,48h时用反转录聚合酶链反应技术测定特异性电泳条带所显示成骨细胞中骨保护蛋白mRNA的相对表达量,酶联免疫法检测48,72和96h时200mL/L终浓度培养上清中骨保护蛋白的浓度。结果:两组动物各10只,均无亡失,所取血清正常用于细胞培养。①成骨细胞在接种4h后可见部分细胞贴壁、伸展。随培养时间延长,细胞伸出较多突起,细胞形态为单核,多边形及梭形,培养2d细胞基本全部贴壁,主要为梭形或多边形,培养6d左右基本贴满瓶壁,呈铺路石样排列。单层铺满培养瓶后可出现重叠生长,传代细胞大约15d天左右开始形成矿化结节。②特异性电泳条带显示益骨胶囊含药血清培养后24h和48h成骨细胞骨保护蛋白mRNA的表达均高于空白血清对照组犤(0.196±0.015,0.236±0.016),(0.152±0.018,0.206±0.022),P<0.01犦;③200mL/L终浓度的益骨胶囊含药血清培养后48,72和96h成骨细胞骨保护蛋白的表达明显高于空白血清对照组犤(0.1230±0.0128,0.1681±0.0130,0.1983±0.0186),(0.1067±0.0106,0.1385±0.0126,0.1588±0.0121),P<0.01或P<0.05犦。结论:益骨胶囊含药血清具有促进成骨细胞表达骨保护蛋白的作用。提示益骨胶囊能够通过对成骨细胞的影响而对骨吸收环节具有抑制作用,但是否对其他信号通路也有作用尚待研究。

AIM:To observe the effects of Yigu capsule drug-containing serum on osteoprotegerin mRNA in rats' osteoblasts cultured in vitro.METHODS:The experiment was carried out in the Cell and Protein Engineering Laboratory,College of Life Science Technology,Jinan University from May 2004 to February 2005.Twenty male 12 month old SD rats were randomly divided into two groups:traditional Chinese medicine (Yigu capsule,which consisted of 8 Chinese herbs,wuch as epimedium,prepared rehmannia,wolfberry fruit,achyranthes root,etc.) group (n=10) and distilled water control group (n=10).The drug containing serum and control serum were prepared with the method of gastric perfusion,and the osteoblasts in skull of new born rats were separated and cultured.After passage,they were randomly divided into drug containing serum group (n=8) and control serum group (n=8),and then the relative expression value of osteoprotegerin mRNA in osteoblasts were determined with the technique of reverse transcription polymerase chain reaction specific electrophoresis strap at 24 and 48 hours of culture respectively,and the concentration of osteoprotegerin in supernatant fluid(end concentration of 200 mL/L) was detected with enzyme linked immunosorbent assay at 48,72 and 96 hours of culture respectively.RESULTS:No rat died in both groups (n=10 respectively),and the taken serum was normally used for cell cultivation.① Four hours after inoculation,stick to wall and extension of osteoblasts were observed.With the prolongation of culture,the osteoblasts extended more processus,the shape was mononuclear,polygon and fusiform shapes;Two days after cultivation,almost all the osteoblasts stuck to wall,the shapes were mainly polygon or fusiform;About 6 days after cultivation,the osteoblasts stuck to almost the complete wall of the bottle,arranged like slabstone;Overlapped growth occurred after the spread of single layer in the culture bottle,the passage cells began to mineralize and nod at about 15 days.② Specific electrophoresis strap showed that the expressions of osteoprotegerin mRNA in osteoblasts 24 and 48 hours after cultivation were higher in the Yigu capsule drug containing serum than in the blank serum[(0.196± 0.015,0.236± 0.016),(0.152± 0.018,0.206± 0.022),P< 0.01].③ The expressions of osteoprotegerin protein in osteoblasts 48,72 and 96 hours after cultivation were obviously higher in the Yigu capsule drug containing serum (end concentration of 200 mL/L) than in the blank serum[(0.123 0± 0.012 8,0.168 1± 0.013 0,0.198 3± 0.018 6),(0.106 7± 0.010 6,0.138 5 ± 0.012 6,0.158 8± 0.012 1),P< 0.01 or P< 0.05].CONCLUSION:Yigu capsule drug containing serum can promote the expression of osteoblasts,indicating that Yigu capsule has the role in inhibiting the link of bone resorption by its influence on osteoblasts,but whether it plays a role in other signal pathways still needs to be investigated.