巴戟天-牛膝醇提物对SD大鼠软骨细胞G_(1/S)周期蛋白与基因表达影响的实验研究  

An Experimental Study on the Effect of Morinda-Achyranthes Alcohol Extract on the Expression of G_(1/S) Cyclin and Gene in Cartilage Cells of SD Rats

作　　者：吴子健[1] 吴桂玲[2] 陈达婷 苏澜 陈春蓉[3] 叶锦霞[4] 洪振强[1] 

机构地区：[1]福建中医药大学,福建福州350122 [2]湖北中医药大学针灸学院,湖北武汉430065 [3]福建中医药大学药学院,福建福州350122 [4]福建中医药大学中西医结合研究院,福建福州350122

出　　处：《风湿病与关节炎》2017年第2期5-9,32,共6页Rheumatism and Arthritis

基　　金：福建省自然科学基金(2016J01779)

摘　　要：目的:探究巴戟天-牛膝醇提物(M-AAE)对体外培养的SD大鼠软骨细胞G_(1/S)周期蛋白与m RNA含量表达的影响,为其防治骨关节炎的临床应用提供依据。方法:1用乙醇加热回流法获得其醇提物成分;采用机械-酶消化法分离SD大鼠膝关节处的关节软骨,建立体外培养细胞模型并进行鉴定;2 MTT法检测M-AAE对软骨细胞增殖特性的影响,使用倒置相差显微镜镜下观察软骨细胞状态;3 PCR、Western blot法分别检测体外培养大鼠软骨细胞经M-AAE的0,75,150,300μg·m L^(-1) DMEM溶液干预48 h后的m RNA、蛋白含量的表达。结果:1软骨细胞经Ⅱ型胶原法染色后,阳性对照组胞浆区域浸染为棕黄色;2经MTT检测M-AAE对软骨细胞增殖具有一定的时效-量效关系,其最佳干预时间和浓度分别为48 h和150μg·m L^(-1);3 M-AAE干预软骨细胞后,各加药组细胞周期蛋白依赖性激酶-4(CDK4)、细胞周期蛋白D1(Cyclin D1)的m RNA与蛋白含量的表达量均明显高于0μg·m L^(-1)组,其中以150μg·m L^(-1)组的表达量最高(P<0.01),75μg·m L^(-1)组和300μg·m L^(-1)组在CDK4及CyclinD1 m RNA上比较,其表达量差异无统计学意义(P>0.05)。结论:M-AEE可上调CDK4、Cyclin D1m RNA及蛋白表达,对SD大鼠软骨细胞具有一定的促增殖作用。Objective：To explore the effect of Morinda-Achyranthes Alcohol Extract （ M-AAE ） on the expression of G1/s cyclin and the content of mRNA in cartilage cells of SD rats,providing the basis for clinical application in the prevention and treatment of osteoarthrifis.Methods： （1） The ethanol refluxing method was used to obtain the alcohol extract components and the mechanical-enzyme digestion method was used to separate articular cartilage from SD rats knee joints to establish models culturing cells in vitro and identify them; （2） The MTT method was used to study the effect of M-AAE on chondrocyte proliferation and the inverted phase contrast microscope was used to observe the state of cartilage cells; （3） The PCR and Western blot methods were used to detect the expression of mRNA and the protein content in cartilage cells of SD rats after 48 h intervention respectively with 0,75,150,300 μg · mL^-1 of DMEM.Results： （1） After type II collagen staining,the cytoplasmic domain of the chondrocyte in the positive control group was brown; （2） By the MTT detection,M-AAE had certainseasoning-dose effect on chondrocyte proliferation,and the best intervention time and concentration were respectively 48 h and 150 μg · mL^-1; （3）After M-AAE intervention, the expressions of mRNA and protein content of CDK4 and Cyclin D1 in drug-increasedgroups were significantly higher than those in the group with 0 μg · mL^-1 of drug,among which the expression of the group with 150 μg · mL^-1 was the highest （P 〈 0.01） .Compared on the aspects of CDK4,Cyclin D1 and mRNA,the expression difference between the group intervened with 75 μg · mL^-1 of drug and that with 300 μg · mL^-1 was not statistically significant （P 〉 0.05） .Conclusion：M-AEE can up-regulate the expressions of CDK4,Cyclin DI,mRNA and protein,which can promote the proliferation of chondrocytes in SD rats.

关 键 词：骨关节炎 巴戟天 牛膝 醇提物 软骨细胞 增殖 大鼠 

分 类 号：R285[医药卫生—中药学]