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
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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 m RNA in cartilage cells of SD rats,providing the basis for clinical application in the prevention and treatment of osteoarthritis.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 m RNA and the protein content in cartilage cells of SD rats after 48 h intervention respectively with 0,75,150,300 μg·m L~(-1) of DMEM.Results: ① After type Ⅱcollagen staining,the cytoplasmic domain of the chondrocyte in the positive control group was brown; ② By the MTT detection,M-AAE had certains easoning- 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 mR NA and protein content of CDK4 and Cyclin D1 in drug-increasedgroups were significantly higher than those in the group with 0 μg·m L~(-1) of drug,among which the expression of the group with 150 μg·m L~(-1) was the highest(P < 0.01).Compared on the aspects of CDK4,Cyclin D1 and m RNA,the expression difference between the group intervened with 75 μg·m L~(-1) of drug and that with 300 μg·m L~(-1) was not statistically significant(P > 0.05).Conclusion:M-AEE can up-regulate the expressions of CDK4,Cyclin D1,mR NA and protein,which can promote the proliferation of chondrocytes in SD rats.
Objective:To explore the effect of Morinda-Achyranthes Alcohol Extract(M-AAE)on the expression of G1/S cyclin and the content of m RNA in cartilage cells of SD rats,providing the basis for clinical application in the prevention and treatment of osteoarthritis.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 m RNA and the protein content in cartilage cells of SD rats after 48 h intervention respectively with 0,75,150,300 μg·m L~(-1) of DMEM.Results: ① After type Ⅱcollagen staining,the cytoplasmic domain of the chondrocyte in the positive control group was brown; ② By the MTT detection,M-AAE had certains easoning- 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 mR NA and protein content of CDK4 and Cyclin D1 in drug-increasedgroups were significantly higher than those in the group with 0 μg·m L~(-1) of drug,among which the expression of the group with 150 μg·m L~(-1) was the highest(P < 0.01).Compared on the aspects of CDK4,Cyclin D1 and m RNA,the expression difference between the group intervened with 75 μg·m L~(-1) of drug and that with 300 μg·m L~(-1) was not statistically significant(P > 0.05).Conclusion:M-AEE can up-regulate the expressions of CDK4,Cyclin D1,mR NA and protein,which can promote the proliferation of chondrocytes in SD rats.
引文
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[11]黄丽婵,杨世奎,刘发元,等.川牛膝、怀牛膝治疗骨关节炎的药效物质基础探讨[J].风湿病与关节炎,2014,3(11):52-54.
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[13]刘发元,党传鹏,翁霞萍,等.牛膝引药下行靶向调节骨关节炎软骨退变的机制探讨[J].风湿病与关节炎,2014,3(6):46-48.
[14]Wu G,Fan H,Huang Y,et al.Duhuo Jisheng Decoction-containing serum promotes proliferation of interleukin-1β-induced chondrocytes through the p16-cyclinD1/CDK4-Rb pathway[J].Mol Med Rep,2014,10(5):2525-2534.
[15]Yu FR,Li X,Cai L,et al.Achyranthes bidentata polysaccharides induce chondrocyte proliferation via the promotion of the G1/S cell cycle transition[J].Mol MedRep,2013,7(3):935-940.
[2]梅阳阳,付长龙,庞书勤,等.乌头汤药理药效研究及其干预骨关节炎的机制探讨[J].风湿病与关节炎,2016,5(6):47-50.
[3]刘献祥.基于陈可冀学术思想之骨性关节炎研究[J].康复学报,2016,26(1):2-5.
[4]袁芳,侯秀娟,刘小平,等.补益肝肾活血通络法治疗膝骨关节炎的理论探讨[J].中华中医药杂志,2016,31(3):891-893.
[5]付长龙,潘彩斌,刘国强,等.药对巴戟天-杜仲总多糖的含量测定及其对大鼠软骨细胞增殖的影响[J].风湿病与关节炎,2014,3(6):23-26.
[6]Liu F,Liu G,Liang W,et al.Duhuo Jisheng decoctiontreatment inhibits the sodium nitroprussiate-inducedapoptosis of chondrocytes through the mitochondrial-dependent signaling pathway[J].Int J MolMed,2014,34(6):1573-1580.
[7]Chen WH,Liu XX,Tong PJ,et al.Diagnosis and management of knee osteoarthritis:Chinese medicine expertconsensus(2015)[J].Chin J Integr Med,2016,22(2):150-153.
[8]郑素玉,陈健.巴戟天有效成分及其药理作用实验研究进展[J].世界中西医结合杂志,2012,7(9):823-825,828.
[9]史辑,崔妮,贾天柱.巴戟天不同炮制品及提取部位抗大鼠佐剂性关节炎的比较研究[J].中药材,2015,38(8):1626-1629.
[10]黄涛,张钢林,李楠.巴戟天多糖对体外培养兔软骨细胞凋亡的影响[J].北京体育大学学报,2010,33(8):56-61.
[11]黄丽婵,杨世奎,刘发元,等.川牛膝、怀牛膝治疗骨关节炎的药效物质基础探讨[J].风湿病与关节炎,2014,3(11):52-54.
[12]林平冬,翁霞萍,刘发元,等.牛膝有效成分防治骨关节炎的作用机制探讨[J].风湿病与关节炎,2015,4(2):56-59.
[13]刘发元,党传鹏,翁霞萍,等.牛膝引药下行靶向调节骨关节炎软骨退变的机制探讨[J].风湿病与关节炎,2014,3(6):46-48.
[14]Wu G,Fan H,Huang Y,et al.Duhuo Jisheng Decoction-containing serum promotes proliferation of interleukin-1β-induced chondrocytes through the p16-cyclinD1/CDK4-Rb pathway[J].Mol Med Rep,2014,10(5):2525-2534.
[15]Yu FR,Li X,Cai L,et al.Achyranthes bidentata polysaccharides induce chondrocyte proliferation via the promotion of the G1/S cell cycle transition[J].Mol MedRep,2013,7(3):935-940.