摘要
目的研究黄芪甲苷对人膝骨关节炎退变关节软骨IL-1β表达的影响。方法取膝骨关节炎患者手术后的退变膝关节软骨,进行细胞培养,将培养的软骨细胞分离传代后,取第三代细胞,通过HE、Ⅱ型胶原免疫荧光染色进行软骨细胞鉴定,并进行IL-1β免疫荧光染色。观察软骨细胞退变情况后分为6组(A组:软骨细胞+DMEM对照组;B组:软骨细胞+25mmol/L黄芪甲苷;C组:软骨细胞+50mmol/L黄芪甲苷;D组:软骨细胞+75mmol/L黄芪甲苷;E组:软骨细胞+100mmol/L黄芪甲苷;F组:软骨细胞+500mmol/L黄芪甲苷),取4、8、24、48、72h采用实时荧光定量聚合酶链式反应(polymerase chainreaction,PCR)检测各组退变软骨细胞内IL-1βmRNA的表达情况。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。IL-1β免疫荧光染色可见清晰绿色荧光,软骨细胞以多角形多见。②IL-1βmRNA表达。不同时间点间软骨细胞IL-1βmRNA表达水平有差异(F=13.236,P<0.01);不同组间软骨细胞IL-1βmRNA表达水平有差异(F=98.762,P<0.01),A组高于B组、C组、D组、E组(P<0.05～0.01),F组高于A组、B组、C组、D组、E组(P<0.05～0.01);时间因素和组间因素存在交互效应(F=7.262,P<0.01)。结论一定浓度的黄芪甲苷能通过抑制退变软骨细胞合成IL-1β,延缓软骨细胞退变;浓度过高则会促进退变软骨细胞合成IL-1β,加速软骨细胞退变。
OBJECTIVE To study the effect of Astragaloside IV on the IL-1βexpression Of degenerative joint cartilage in human knee osteoarthritis（OA）. METHODS Degenerative joint cartilage cells of postoperative knee OA patients were isola- ted, cultured and passaged in vitro. The third generation cells were identified by HE and type Ⅱ collagen immunofluorescence staining, and then were stained by IL-1βimmunofluorescence. They were divided into 6 groups after the cell degenerative situ- ation was observed （Group A： cartilage cells ＋ DMEM control group; Group B： cartilage cells ＋ 25 mmol/L Astragaloside IV; Group C： cartilage cells ＋ 50 mmol/L Astragaloside IV; Group D： cartilage cells ＋ 75 mmol/L Astragaloside IV; Group E： cartilage cells ＋ 100 mmol/L Astragaloside IV; Group F： cartilage cells ＋ 500mmol/L Astragaloside IV）,, and real-time fluorescent quantitative PCR wassed to detect IL-1 βexpressing mRNA in degenerative cartilage cells of every group in 4 h, 8 h, 24 h,48 h and 72 h. RESULTS （1）Morphologic observation. Spindle cells and polygonal cells were found in the initial gen- eration cells at the same time. Nucleus located at the side of the cell, closing to the membranes, and the cell filaments were long. HE stained the cytoplasm into red and the membranes into blue. Toluidine blue stained the cytoplasm and membranes in-to dark blue. The positive type Ⅱ collagen stain was green fluorescence. The nucleus were testained by 4＇,6-diamidino-2-phe- nylindole. Activated by fluorescence in various wave bands, the plasm and membranes presented clear green fluorescence, while the nucleus areas showed no obvious green fluorescence, which proved that type 11 collagen mainly distribute in plasm and membranes, and the cultured cells were cartilage cells indeed. The clear green fluorescence could .be seen after IL-1 immu- nofluorescence staining, and polygonal cells were dominated among cartilage cells. （2）IL-1 mRNA expression. The IL-1 mRNA expression levels in different time showed difference （F= 13. 236, P〈0.01）; and those in different grotlps were different as well, Group A higher than Group B, C, D, E （P〈0. 05--0. 01） and Group F higher than Group A, B, C, D, E （P〈0.05~ 0.01）; the factors of time and group had interaction effect （F= 7. 262, P〈0.01）. CONCLUSION Astragaloside IV with a certain concentration can delay the degeneration of cartilage cells by inhibiting IL-1 synthesis in the degenerative cartilage cells, but it with a excessive high concentration will speed up the degeneration by promoting IL-1 synthesis.
出处
《南京中医药大学学报》
CAS
CSCD
北大核心
2013年第1期48-52,共5页
Journal of Nanjing University of Traditional Chinese Medicine
基金
江苏省中医药局科研计划项目(HZ07089)
关键词
膝骨关节炎
退变
黄芪甲苷
软骨细胞
IL-1Β
knee osteoarthritis
degeneration
Astragaloside IV
cartilage cells
IL-1β
