威灵仙干预体外培养兔膝关节软骨细胞增殖及转化生长因子β1 mRNA基因的表达

Effect of Weilingxian on proliferation of rabbit knee articular chondrocytes cultured in vitro and mRNA expression of transforming growth factor-beta 1

背景:有研究显示,威灵仙能够维持和促进软骨细胞合成蛋白多糖与Ⅱ型胶原,并可能通过抑制白细胞介素1水平的增高保护关节软骨,延缓骨关节炎的发展。目的:在以往研究基础上,观察中药威灵仙对体外培养兔膝关节软骨细胞增殖及转化生长因子β1mRNA基因表达的影响,探讨其治疗骨关节炎的作用及可能机制。方法:取新西兰白兔膝关节软骨,剪碎后,采用酶消化法原代分离培养软骨细胞,甲苯胺蓝染色鉴定后,取处于对数生长期第3代细胞随机分组,实验组分别加入0.01,0.05,0.1,0.5,1.0g/L威灵仙培养基,对照组仅加入普通培养基进行培养。倒置显微镜下观察原代培养的软骨细胞形态变化及甲苯胺蓝染色鉴定,MTT法检测不同质量浓度威灵仙对软骨细胞增殖影响,RT-PCR法检测转化生长因子β1mRNA表达。结果与结论:原代软骨细胞呈圆球形,悬浮状态,24h后大部分细胞贴壁;传代后软骨细胞生长速度加快,融合成片,外观呈典型"铺路石"状;传4代后逐渐出现长梭形软骨细胞,传代培养至6代时,呈成纤维细胞样外观,生长速度明显减慢,传代周期延长。软骨细胞经甲苯胺蓝染色后细胞外基质可见蓝色异染颗粒,细胞核染成深蓝色。不同质量浓度威灵仙均能促进软骨细胞增殖,尤以0.5g/L增殖效果明显,且增殖高峰出现在威灵仙干预后的第3天。0.05,0.1,0.5,1.0g/L威灵仙均能促进软骨细胞转化生长因子β1mRNA表达,4组组间比较差异无显著性意义(P>0.05),但作用的高峰为0.5g/L组。威灵仙能促进软骨细胞增殖及转化生长因子β1mRNA的表达,提示这可能是其治疗骨关节炎的作用及可能机制之一。

BACKGROUND： Studies have shown that Weilingxian can maintain and promote the synthesis of proteoglycan and collagen Ⅱ of chondrocyte, and protect articular cartilage and postpone the development of osteoarthritis by inhibiting the level of interleukin-1 （IL-1） possibly. OBJECTIVE： Based on the previous studies, to observe the effect of Weilingxian on the proliferation of rabbit knee articular chondrocyte and transforming growth factor-β1 mRNA expression, and then to explore the role and possible mechanism of Weilingxian in the treatment of osteoarthritis. METHODS： Knee cartilage was shredded after harvested from New Zealand white rabbits under sterile conditions, and chondrocytes were isolated and cultured by the way of enzymatic digestion. After identifying by toluidine blue staining, the third-passage cells in the logarithmic growth phase were cultured in vitro and randomly divided into two groups after adherence. The experimental groups were cultured in DMEM with 0.01, 0.05, 0.1, 0.5, and 1.0 mg/mL Weilingxian, while the control group was given with normal medium alone. Chondrocytes morphology was observed under an inverted phase contrast microscope, and the phenotype was identified by toluidine blue staining; Methyl Thiazolyl Tetrazolium （MTT） assay method was adopted to observe the influence of Weilingxian with different concentrations on the proliferation of chondrocytes, and anti-transcription-polymerase chain-type reaction （RT-PCR） was used to assay the expression changes of transforming growth factor-β1 mRNA. RESULTS AND CONCLUSION： Primary cultured chondrocyte was round-shaped, and most of it adhered after 24 hours, the appearance was polygonal and irregular-shaped; after passage, cell growth was faster than before, the typical appearance was slabstone-like; long spindle-shaped chondrocytes appeared after four generations; after six generations, most cells showed long spindle-shaped fibroblast-like appearance, the rate of growth also slowed down. Extracellular matrix of chondrocytes was stained to be blue by toluidine blue staining, and the nucleus was dark blue. Different concentrations of Weilingxian could promote the proliferation of chondrocytes, effect of 0.5 mg/mL group was significantly, and the peak of proliferation was on the third day. 0.05, 0.1, 0.5, and 1.0 mg/mL Weilingxian group could promote the expression of transforming growth factor-β1 mRNA, and there was no significant difference between four groups （P 〉 0.05）, but the peak was at 0.5 mg/mL group. Weilingxian can promote proliferation of chondrocyte and transforming growth factor-β1 mRNA expression, and these may be one of the possible mechanisms that Weilingxian can work in the treatment of osteoarthritis.