“补肾益肝活血方”对小鼠骨髓基质细胞成软骨分化相关基因表达的影响

The Impact of Related Gene Expression of the Chondrogenic Differentiation of Rat Bone Marrow Stromal Cells by Bu-Shen-Yi-Gan-Huo-Xue-Decoction

目的:采用血清药理学方法,从基因水平探索中药补肾益肝活血方影响小鼠骨髓基质细胞(BMSCs)成软骨细胞分化的机制。方法:取45只昆明大鼠随机分为3组,补肾益肝活血方高、低剂量组分别以含14.67×4g生药/kg、14.67g生药/kg的水溶液2mL灌胃,对照组以等量生理盐水2mL灌胃,2次/d,连续给药3d后,腹主动脉采血制备血清。另从4只昆明小鼠骨髓中分离出BMSCs,贴壁法培养传代,取第3代BMSCs分为4组:对照组、阳性组及高、低剂量中药血清诱导组,依次以空白血清、成软骨诱导阳性药及高、低剂量中药血清诱导BMSCs成软骨细胞分化。苏木精-伊红染色观察各组软骨分化情况,基因芯片技术分析成软骨分化基因表达差异谱,并用Real time PCR验证。结果:苏木精-伊红染色显示阳性组及高、低剂量中药血清诱导组均能促进BMSCs成软骨细胞分化。阳性组及高、低剂量中药血清诱导组中软骨细胞相关的金属基质蛋白酶-3(MMP3)、含纤蛋白样表皮生长因子细胞外基质蛋白-1(EFEMPⅠ)均为下调基因(下调倍数>2)。Real time PCR示阳性组及高、低剂量中药血清诱导组中Ⅱ型胶原(CollagenⅡ)、SOX9基因及糖胺聚糖(Aggrecan)的表达均增高,表明三组均有软骨生成,且MMP3、EFEMP1的表达下降,进一步验证了基因芯片结果。结论:补肾益肝活血方含药血清能有效诱导BMSCs成软骨细胞分化,其作用机制很可能与MMP3、EFEMP1介导的通路有关。

Objective：To explore the effects of Bu-Shen-Yi-Gan-Huo-Xue-Decoction （BSYGHXD） on the related gene ex- pression of chondroblastic differentiation of rat bone marrow stromal cells and the mechanisms involved. Methods： BSYGHXD （including Radix Salviae Miltiorrhizae, Rhizoma Drynariae, Myrrha, Radix Achyranthis Bidentatae） containing serum was prepared by gavage administration of the decoction to the rats at a dose of 14.67×4g/kg （high dose）, 14.67g/kg （low dose） twice a day for 3 days, and control group with NS. Bone marrow stromal cells were isolated from the femoral and tibial marrow of rats and were cultured in vitro. The third generation of cells were taken into chondroblastie differentia- tion experiments, induced by 1% v/v ITS ＋ TGF-β1 10ng/ml ＋ DEXA 100nM ＋VitC 50μg/ml and BSYGHXD contai- ning serum. Morphology of chondroeytes were observed by HE staining, Gene expression was carried out by gene chip, and Real Time-PCR was performed to detect the expression of Collagen Ⅱ, SOX9, Aggrecan and novel genes found by gene chip. Results：The results of HE staining were positive in positive group and BSYGHXD containing serum inducing groups. Expression of matrix metalloproteinase 3 （MMP3） and epidermal growth factor-containing fibulin-like extracellular matrix protein 1 （EFEMP1） related to chondroblastic differentiation was down-regulated （≤0.5fold）, and results of Real Time- PCR confirmed overexpression of Collagen II, SOX9 and Aggrecan and down-regulated expression of MMP3 and EFEMP1. Conclusion：BSYGHXD can induce bone marrow stromal cells into chondrocytes, and possibly be mediated by MMP3 or EFEMP1 pathway.