益气化瘀补肾方联合TGF-β1诱导大鼠BMSCs向类髓核细胞分化的研究

Effect on Differentiation of BMSCs into Nucleus Pulposus Cells by Yiqi Huayu Bushen Recipe combined with TGF-β1 in Rats

目的:观察中药益气化瘀补肾方含药血清联合转化生长因子β1(transforming growth factor-β1,TGF-β1)体外诱导大鼠骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)向类髓核细胞分化的效果,探讨益气化瘀补肾方在协同诱导、调节分化方面的作用。方法:取成年大鼠股骨骨髓,贴壁培养法获得原代BMSCs,体外培养、分离及纯化。取第3代大鼠BMSCs,分为空白对照组(A组)、单纯中药含药血清诱导组(B组)、单纯TGF-β1诱导组(C组)以及中药含药血清联合TGF-β1诱导组(D组)。A组加入含10%胎牛血清(FBS)的L-DMEM培养基继续培养,B组为含10%益气化瘀补肾方含药血清的L-DMEM培养基,C组为含10μg/L TGF-β1的LDMEM培养基,D组为含10%益气化瘀补肾方含药血清及10μg/L TGF-β1的L-DMEM培养基。连续培养3、7、14、21 d,采用倒置相差显微镜进行细胞形态学观察,使用Alcian Blue染色检测蛋白多糖表达情况,免疫组化法检测Ⅱ型胶原表达情况,使用RT-PCR方法检测蛋白多糖及Ⅱ型胶原m RNA表达情况。结果:培养3 d时,仅有D组可检出蛋白多糖和Ⅱ型胶原表达。培养至第14天时,D组蛋白多糖、Ⅱ型胶原及其m RNA水平明显高于其它实验组(P<0.05)。结论:TGF-β1可以诱导大鼠BMSCs向类髓核细胞分化,联合中药益气化瘀补肾方含药血清能进一步提高诱导分化的效率,揭示了中药在干细胞组织工程领域的潜在作用。

Objective： To investigate the effect on differentiation of BMSCs into nucleus pulposus cells by Yiqi Huayu Bushen Recipe combined with TGF-β1 in rats. Method： Adult rat bone marrow was obtained from the femoral bone marrow of adult rat. The BMSCs was obtained by adherent culture method. The third generation rat BMSCs were selected and divided into the blank control group（ group A）,the pure Chinese medicine containing serum induced group（ group B）,the pure TGF-β 1 induced group（ group C） and the drug containing serum with TGF- β 1 induced group（ group D）. The group A with 10% fetal calf serum（ FBS） was added to the L- DMEM group. The group B contained 10% Yiqi Huayu Bushen Recipe serum containing L-DMEM medium. The group C was L-DMEM medium containing 10 μ g / L TGF-β1.The D group is L-DMEM medium containing 10% Yiqi Huayu Bushen Recipe serum and 10 μ g / L TGF- β 1.Continuous culture 3,7,14,21 days,the cell morphology was observed by inverted phase contrast microscope,the blue Alcian staining was used to detect the protein polysaccharide,the expression of type II collagen was detected by immunohisto-chemical techniques,the expression of m RNA aggrecan and collagen m RNA was detected by RT-PCR method. At 3 days,only the D group could be detected the expression of protein polysaccharide and collagen type II. And at 14 days,the D group was significantly higher than other experimental group in expression of protein polysaccharide and collagen type II and their m RNA expression（ P〈0.05）.Conclusion： TGF-1 can induce rat BMSCs to the nucleus pulposus cell differentiation,combined with Yiqi Huayu Bushen recipe medicated serum can significantly improve efficiency of induced differentiation,and the experimental results further reveal the potential role of traditional Chinese medicine in the field of stem cell tissue engineering.