六味地黄汤含药血清对兔软骨细胞表型的影响

目的:探讨六味地黄汤含药血清对关节软骨细胞增殖及表型的影响。方法:无菌条件分离新生24小时胎兔长骨干骺端透明软骨,用酶消化法分离原代关节软骨细胞,取P_2代软骨细胞分别种于96孔板(用于细胞增殖实验)和6孔板(用于细胞分泌性蛋白检测),设置空白对照组、正常兔血清组和六味地黄汤含药血清组,每组4个复孔。连续干预96小时后,CCK8法测定细胞增殖情况,ELISA法测定各组培养上清中Ⅱ型胶原(CollagenⅡ,colⅡ)、糖胺聚糖(glycosaminoglycan,GAG)含量。结果:干预结束后,CCK8法测定正常兔血清组和六味地黄汤含药血清组OD值均高于空白对照组,六味地黄汤含药血清组OD值高于正常兔血清组,差异均具有统计学意义(P0.05);ELISA法检测六味地黄汤含药血清组colⅡ、GAG含量均高于空白对照组,差异均具有统计学意义(P0.05);与正常兔血清组比较,六味地黄汤含药血清组GAG含量高于正常兔血清组,差异具有统计学意义(P0.05)。结论:六味地黄汤含药血清可以促进体外培养兔关节软骨细胞的增殖,促进软骨细胞分泌Ⅱ型胶原和GAG,从而更好地维持软骨细胞表型。

Effect of Serum Containing LiuWei DiHuang Decoction on Chondrocyte Phenotype of Rabbit

Objective:To study the effect of serum containing LiuW ei DiHuang Tang on proliferation and phe-notype of articular chondrocytes. Methods:Metaphysis of hyaline cartilage of 24-hour new born rabbit was separated under aseptic condition, enzyme digestion method was used to isolate the primary generation chondrocyte of joint cartilage, P2 generation chondrocyte of joint cartilage was chosen to be cultured in 96-well plates (for cell prolifera-tion test) and 6-well plates (for cell secretory protein detection). The blank control group, normal rabbit serum group and serum containing LiuWei DiHuang Tang group were set up, four complex holes were included in each group. Cellular proliferation condition was tested by CCK8 method after 96-hour continuous intervention. ELISA method was adopted to test the content of collagenⅡ (colⅡ) and glycosaminoglycan (GAG) in the supernatant of each group. Results:By the end of the intervention, OD values tested by CCK8 of the normal rabbit serum group and the serum containing LiuWei DiHuang Tang group were higher than those of the blank control group, and the OD value of the serum containing LiuW ei DiHuang Tang group was higher than that of the normal rabbit serum group, the dif-ferences were statistically significant (P<0.05). ColⅡ and GAG content of the serum containing LiuWei DiHuang Tang group tested by ELISA method both were higher those of the blank control group, the differences were both statistically significant (P<0.05). Compared with the normal rabbit serum group, GAG content of the serum contain-ing LiuWei DiHuang Tang group was higher, the differences was statistically significant (P<0.05). Conclusion:Serum containing LiuW ei DiHuang Tang can improve the proliferation of the rabbit articular chondrocytes in vitro and increase the secretory content of collagenⅡ and glycosaminoglycan (GAG) of the chondrocyte, therefore, to maintain the phenotype of chondrocyte well.