微应变环境下柚皮苷对骨髓间充质干细胞成骨分化的影响

Effect of naringin monomer on osteogenic differentiation of bone mesenchymal stem cells under microstrain environment

目的探讨在微应变环境下,柚皮苷体外对培养的骨髓间充质干细胞(MSCs)向成骨细胞分化的影响及其机制。方法将体外培养的兔MSCs接种于硅橡胶膜上,利用EF3200力学试验仪搭载的BioDynamic生物反应舱系统,对细胞进行周期性循环牵张微应变(微应变)加载。实验分成8组:对照组(常规培养),微应变组,柚皮苷不同质量浓度(2、20、200 ng/mL)组,微应变+柚皮苷(2、20、200 ng/mL)组。流式细胞仪检测各组MSCs的增殖指数;RT-PCR检测各组MSCs骨钙蛋白(OCN)、成骨特异性转录因子(Runx 2)与I型胶原(Col I)基因的表达。结果在微应变环境下,柚皮苷使MSCs呈现明显的极性排列趋向,且细胞长轴平行力学刺激方向;显著提高MSCs的增殖活性;柚皮苷200 ng/mL显著上调OCN基因表达;不同质量浓度的柚皮苷均显著上调Runx 2基因表达,且与质量浓度呈正相关;低质量浓度柚皮苷促进Col I基因表达,而高质量浓度柚皮苷抑制了Col I基因的表达。结论柚皮苷可增强在微应变环境下MSCs的增殖并能够促进其向成骨分化。

Objective Under the microstrain environment, to investigate the impact and mechanisms of osteoblast differentiation of the naringin monomer on marrow stem cells （MSCs） in vitro. Methods The cyclical stretch microstrain was loaded on silicone rubber membrane with cultured rabbit MSCs using EF3200 mechanical tester with the system of BioDynamic biological reaction tank. The experiment was divided into eight groups, A： the blank control group in conventional cultured environment; B： the group of alone microstrain loading; C： the group cultured in environment containing naringin and the naringin concentrations, respectively were 2, 20, and 200 ng/mL; D： under microstrain environment, the joint application with naringin at concentration of 2, 20, and 200 ng/mL. Flow cytometry was used to test the proliferation index （PI） of MSCs after microstrain loading. The gene expression of the cells in osteocalcin （OCN）, osteoblast specific transcription factor （Runx2） and collagen type I （Col I） were assayed by RT-PCR. Results Under the microstrain environment combined with naringin, MSCs showed obvious polarity and the long axis of cells parallel to the direction of mechanical stimulus direction. The combination of naringin and microstrain stimulation can significantly improve the MSCs proliferation activity. Naringin （200 ng/mL） could improve the gene expression of OCN under the stimulation of 50 000 microstrain. Under 50 000 microstrain stimulation, naringin at different concentration could significantly enhance the Runx2 gene expression and the effect between the enhancement and the naringin concentration was positive. Under the microstrain stimulation, naringin at low concentration could promote the Col I gene expression, on the contrary, naringin at high concentration could inhibit theCol I gene expression. Conclusion Under the microstrain environment, naringin monomer could enhance the proliferation of MSCs and promote the osteogenetic differentiation of MSCs.