黄芪甲苷对人膝骨关节炎退变关节软骨细胞基质金属蛋白酶-1及基质金属蛋白酶-3mRNA表达的影响  

Effect of astragaloside Ⅳ on the expression of matrix metalloproteinase-1 mRNA and matrix metalloproteinase-3 mRNA in the chondrocytes of degenerated joint for patients with knee osteoarthritis

作　　者：孙书龙[1] 姜宏[2] 汤晓晨[2] 孟祥奇[2] 龚正丰[2] 王拥军[3] 莫文[4] 

机构地区：[1]南京中医药大学,江苏南京210046 [2]江苏省苏州市中医医院,江苏苏州215009 [3]上海中医药大学脊柱病研究所,上海200032 [4]上海中医药大学附属龙华医院,上海200032

出　　处：《中医正骨》2012年第10期5-9,共5页The Journal of Traditional Chinese Orthopedics and Traumatology

基　　金：江苏省中医药管理局计划项目(HZ07089)

摘　　要：目的:研究黄芪甲苷对人膝骨关节炎退变关节软骨细胞基质金属蛋白酶-1及基质金属蛋白酶-3mRNA表达的影响。方法:取膝骨关节炎患者膝关节软骨进行细胞培养,将培养的软骨细胞分离传代后取第3代细胞,通过HE染色、甲苯胺蓝染色和Ⅱ型胶原免疫荧光染色进行软骨细胞鉴定,并进行基质金属蛋白酶-1和基质金属蛋白酶-3免疫荧光染色。观察软骨细胞退变情况后将其分为6组,A组不进行干预;B组加入25 mmol.L-1黄芪甲苷;C组加入50 mmol.L-1黄芪甲苷;D组加入75 mmol.L-1黄芪甲苷;E组加入100 mmol.L-1黄芪甲苷;F组加入500 mmol.L-1黄芪甲苷。分别于培养开始后4 h、8 h、24 h、48 h、72 h 5个时间点,采用实时荧光定量聚合酶链式反应检测各组退变软骨细胞内基质金属蛋白酶-1和基质金属蛋白酶-3mRNA表达的情况。结果:①形态观察。原代细胞中梭形细胞和多角形细胞并存,细胞核偏于细胞的一侧而贴近细胞膜,细胞纤丝较长。HE染色细胞质为红色,胞膜呈蓝色;甲苯胺蓝染色细胞质和胞膜均呈深蓝色;Ⅱ型胶原染色阳性为绿色荧光,细胞核采用4’,6-二脒基-2-苯基吲哚复染,在不同波段荧光激发下,胞浆和胞膜可见清晰绿色荧光,细胞核区域未见明显绿色荧光,证明培养细胞表达特征性基因Ⅱ型胶原,主要分布在胞浆和细胞膜上,证明所培养的细胞为软骨细胞。基质金属蛋白酶-1和基质金属蛋白酶-3免疫荧光染色可见清晰绿色荧光,软骨细胞以多角形多见。②基质金属蛋白酶-1mRNA表达。不同时间点间软骨细胞基质金属蛋白酶-1mRNA表达水平有差异(F=11.027,P=0.000);不同组间软骨细胞基质金属蛋白酶-1mRNA表达水平有差异(F=102.345,P=0.000),A组高于B组、C组、D组、E组(P=0.000;P=0.000;P=0.000;P=0.000),F组高于A组、B组、C组、D组、E组(P=0.009;P=0.000;P=0.000;P=0.000;P=0.000);时间因素和组间因素存在交互效应(F=8.258,P=0.000)。③�Objective：To study on the effect of astragaloside Ⅳ on the expression of matrix metalloproteinase-1 mRNA（MMP-1 mRNA）and matrix metalloproteinase-3 mRNA（MMP-3 mRNA）in the chondrocytes of degenerated joint for patients with knee osteoarthritis（KOA）.Methods：Knee joint cartilages were fetched out from KOA patients for cell culture,and the third generation cells were chosen from the cultured chondrocytes after isolation and subculture.Chondrocytes identification was made through HE staining,toluidine blue staining and collagen typeⅡimmunofluorescence staining,then immunofluorescent staining was used to detect MMP-1 and MMP-3.The chondrocytes were divided into 6 groups after observing their degeneration conditions.Cells in group A were not intervened,cells in other groups were placed in the culture fluids respectively added with 25 mmol·L-1 astragaloside Ⅳ（group B）,50 mmol·L-1 astragalosideⅣ （group C）,75 mmol·L-1 astragalosideⅣ（group D）,100 mmol·L-1 astragalosideⅣ（group E）,and 500 mmol·L-1 astragalosideⅣ（group F）.The expression of MMP-1 mRNA and MMP-3 mRNA in the degenerated chondrocytes of the 6 groups were inspected through real-time fluorescence quantitative polymerase chain reaction 4,8,24,48 and 72 hours after the culture respectively.Results：①Morphological observations：the situations as coexistence of spindle cells and polygonal cells,the nucleus located at the side of cells were close to the cell membrane and the longer cell microfibril were shown in primary cells.Red cytoplasm and blue cell membrane were shown after HE staining;cytoplasm and cell membrane were dark blue after toluidine blue staining,collagen typeⅡ-positive cells were shown of green fluorescence after immunofluorescence staining,and nucleus were further processed with counterstaining through 4＇,6-diamidino-2-phenylindole,then the clear green fluorescence was shown in cytoplasm and cell membrane under fluorescence excitation in different wavebands,while there was not obvious green f

关 键 词：骨关节炎 膝 软骨细胞 黄芪甲苷 基质金属蛋白酶1 基质金属蛋白酶3 

分 类 号：R274.9[医药卫生—中西医结合]