论文部分内容阅读
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 observe the effects of Yiqi Huayu Bushenfang serum combined with transforming growth factor-β1 (TGF-β1) induced bone marrow-derived mesenchymal stem cells (BMSCs) Cell differentiation, to explore the Qi and blood stasis Bushen Fang in the synergistic induction, regulation of differentiation in the role. Methods: BMSCs were harvested from adult rat femur and cultured in vitro. Primary cultured BMSCs were isolated, purified and purified. The third generation of rat BMSCs were divided into blank control group (A group), simple Chinese medicine-containing serum induction group (B group), simple TGF-β1 induction group (C group) and traditional Chinese medicine serum combined with TGF- Group (Group D). Group A was cultured in L-DMEM containing 10% fetal bovine serum (FBS), Group B was L-DMEM containing 10% Yiqi Huayu Bushen Recipe containing serum, group C containing 10μg / L TGF-β1 in LDMEM medium, and group D was L-DMEM medium containing 10% Yiqi Huayu Bushen Fang containing serum and 10 μg / L TGF-β1. The cells were cultured for 3, 7, 14 and 21 days. The morphology of the cells was observed by inverted phase contrast microscope. The expression of proteoglycan was detected by Alcian Blue staining. The expression of type Ⅱ collagen was detected by immunohistochemistry. And type Ⅱ collagen m RNA expression. Results: After 3 days of culture, proteoglycan and type Ⅱ collagen were detected only in group D. At the 14th day, the levels of proteoglycan D, collagen Ⅱ and m RNA in group D were significantly higher than those in other experimental groups (P <0.05). Conclusion: TGF-β1 can induce the differentiation of rat BMSCs into nucleus pulposus cells. Combined with Chinese medicine Yiqi Huayu Bushen Recipe containing serum, the potential of inducing differentiation can be further enhanced and the potential role of traditional Chinese medicine in the field of stem cell tissue engineering has been revealed.