加味阳和汤含药血清联合转化生长因子β1对骨髓间充质干细胞成软骨分化的影响

Effect of Jiawei Yanghe Decoction containing serum combined with transforming growth factorβ1 on chondrogenic differentiation of bone marrow mesenchymal stem cells

目的探究加味阳和汤含药血清联合转化生长因子β1(TGF-β1)对大鼠骨髓间充质干细胞(BMSCs)成软骨分化的影响。方法全骨髓贴壁培养法分离培养SD大鼠BMSCs,并进行流式细胞术鉴定;CCK-8实验筛选出加味阳和汤含药血清促进BMSCs增殖的最佳有效浓度;将第3代BMSCs分为空白对照组、加味阳和汤含药血清组(含药血清组)、TGF-β1组(阳性对照组)、加味阳和汤含药血清联合TGF-β1组(联合组),诱导分化21 d后,阿利新蓝染色检测软骨特异性基质蛋白糖胺聚糖生成;RT-PCR和蛋白免疫印迹法(Western blot)检测软骨分化标记基因SOX-9、Ⅱ型胶原(ColⅡ)、聚集蛋白聚糖(Aggrecan)的基因蛋白表达。结果①阿利新蓝染色显示,空白对照组未见明显软骨特异性基质蛋白糖胺聚糖(呈蓝色)产生,其余3组可见明显糖胺聚糖生成,其中联合组效果最佳;②与空白对照组比较,Aggrecan、ColⅡ及SOX-9的基因蛋白表达均明显升高(P<0.05),其中联合组效果最佳(P<0.01),且明显优于含药血清组和阳性对照组。结论加味阳和汤含药血清在一定浓度范围内可以促进大鼠BMSCs增殖;加味阳和汤含药血清可以诱导大鼠BMSCs成软骨细胞分化,其与TGF-β1联合使用具有协同促进作用。

Objective To explore the effect of Jiawei Yanghe Decoction(JWYHD)containing serum combined with TGF-β1 on chondrogenic differentiation of rat BMSCs.Methods BMSCs of SD rats were isolated and cultured by whole bone marrow adherent culture and identified by flow cytometry.The best effective concentration of serum containing JWYHD to promote the proliferation of BMSCs was screened by CCK-8 experiment.The third generation of BMSCs was divided into blank control group,JWYHD containing serum group(drug-containing serum group),TGF-β1 group(positive control group),JWYHD containing serum group combined with TGF-β1 group(combined group).After 21 days of induced differentiation,cartilage specific matrix pro⁃tein glycosaminoglycan production was detected by Alcian blue staining.RT-PCR and Western blotting were used to detect the gene protein expression of cartilage differentiation marker genes SOX-9,ColⅡand Aggrecan.Results①Alcian blue staining showed that there was no significant cartilage specific matrix protein glycosaminoglycan(blue)production in the blank control group,but obvious glycosaminoglycan production could be seen in the other three groups,among which the combination group had the best effect.②Compared with the blank control group,the gene protein expressions of Aggrecan,Col II and SOX-9 were sig⁃nificantly increased(P<0.05),and the effect of the combined group was the best(P<0.01),and was significantly better than that of the drug-containing serum group and the positive control group.Conclusion The drug-containing serum of JWYHD can promote the proliferation of rat BMSCs in a certain concentration range;The drug-containing serum of JWYHD can induce the differentiation of rat BMSCs chondroblasts,and its combination with TGF-β1 has a synergistic effect.