脂肪干细胞成骨诱导衍生的外泌体对成骨细胞增殖与矿化的影响

Effects of adipose stem cell osteogenesis-induced exosomes on osteoblast proliferation and mineralization

目的:探讨脂肪干细胞成骨诱导过程中衍生的外泌体对成骨细胞增殖与矿化的影响。方法:从SD大鼠腹部提取脂肪干细胞,将细胞分为两组,一组通过细胞转染沉默细胞内的miR-34a-5p基因,另一组不做处理。两组细胞均使用成骨诱导培养基培养14 d,随后提取细胞分泌在培养液中的外泌体。从SD大鼠幼鼠头盖骨提取成骨细胞,分为对照组、观察组A、观察组B三组。对照组不做任何干预,观察组A给予正常外泌体干预,观察组B给予miR-34a-5p沉默外泌体干预。细胞干预培养7 d后通过细胞增殖实验、碱性磷酸酶染色、茜素红染色和成骨分化基因PCR实验评估脂肪干细胞外泌体对成骨细胞增殖和分化的影响及miR-34a-5p的作用。结果:与对照组和观察组B相比,观察组A成骨细胞数量明显升高,差异有统计学意义(P<0.05);对照组和观察组B比较,差异无统计学意义(P>0.05)。与对照组和观察组B相比,观察组A碱性磷酸酶染色、茜素红染色更深,差异有统计学意义(P<0.05);对照组和观察组B比较,差异无统计学意义(P>0.05)。与对照组和观察组B相比,观察组A成骨分化基因表达更高,差异有统计学意义(P<0.05);对照组和观察组B比较,差异无统计学意义(P>0.05)。结论:脂肪干细胞成骨诱导过程中衍生的外泌体通过miR-34a-5p促进成骨细胞增殖与矿化。

Objective To investigate the effect of exosomes produced during the osteogenic differentiation of adipose derived stem cells on the proliferation and mineralization of osteoblasts.Methods Adipose stem cells were extracted from the abdomen of SD rats.The cells were divided into two groups.One group was transfected with miR-34a-5p gene to silence the cells,and the other group was not treated.The two groups of cells were cultured in osteogenic induction medium for 14 days,and then the exosomes secreted by the two groups of cells in the culture medium were extracted.Osteoblasts were extracted from the skulls of SD rats and divided into control group,observation group A and observation group B.The control group was not done any intervention;Observation group A was treated with exosomes secreted by normal cells;The observation group B was treated with exosomes secreted by miR-34a-5p silenced cells.After 7 days of cell intervention and culture,the effects of exosomes produced in the osteogenic differentiation of adipose stem cells on the proliferation and differentiation of osteoblasts were evaluated by cell proliferation test,alkaline phosphatase staining,alizarin red staining and osteogenic differentiation gene PCR test,and the role of miR-34a-5p was analyzed.Results Compared with control group and observation group B,the number of osteoblasts in observation group A increased significantly(P<0.05);There was no significant difference between control group and observation group B(P>0.05).Compared with control group and observation group B,alkaline phosphatase staining and alizarin red staining in observation group A were deeper(P<0.05);There was no significant difference between control group and observation group B(P>0.05).Compared with control group and observation group B,the expression of osteogenic differentiation gene in observation group A was higher(P<0.05);There was no significant difference between control group and observation group B(P>0.05).Conclusion The exosomes produced during the osteogenic differentiation of adipose derived stem cells can promote the proliferation and mineralization of osteoblasts through miR-34a-5p.