载microRNA-15b-5p壳聚糖纳米粒子的制备及其对大鼠BMSCs成骨分化影响的初步研究

Preparation of MicroRNA-15b-5p Chitosan Nanoparticles and Preliminary Study of the Effects on Osteogenic Differentiation of Rat BMSCs

目的:探讨microRNA(miR)-15b-5p对大鼠来源骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化的调控功能,并构建壳聚糖/miR-15b-5p纳米粒子,研究该纳米粒子对于大鼠BMSCs成骨分化的影响。方法:提取大鼠原代BMSCs并诱导其成骨分化,检测miR-15b-5p在成骨分化过程中的表达变化。再在大鼠BMSCs中过表达miR-15b-5p并诱导其成骨分化,检测其成骨分化能力。进一步采用离子交联法制备不同质量比的壳聚糖/miR-15b-5p纳米粒子并表征其性能,将纳米粒子体外转染BMSCs及体内应用后,检测其成骨分化能力。其中,对照组不经任何处理,使用成骨分化诱导的为诱导组,过表达miR-15b-5p并成骨诱导的为转染诱导组。结果:在大鼠BMSCs成骨分化过程中,miR-15b-5p表达上调;过表达miR-15b-5p并诱导成骨后,其成骨相关基因(RUNX2、OPN、OCN等)表达高于诱导组和对照组;当壳聚糖/miR-15b-5p质量比为20∶1时,纳米粒子包封率达到(98.55±0.10)%,其形态均一,平均粒径为(156.6±9.0)nm,表面电荷为(47.0±0.5)mV。荧光检测显示壳聚糖/miR-15b-5p纳米粒子成功进入胞质。体内、外试验显示运用纳米粒子后,BMSCs成骨分化能力提高。结论:miR-15b-5p能够有效促进大鼠BMSCs成骨分化;使用壳聚糖纳米粒子负载miR-15b-5p能够促进大鼠BMSCs成骨分化及体内骨缺损修复。

Objective:To explore the regulatory function of miR-15b-5p on osteogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs)and construct chitosan/miR-15b-5p nanoparticles to investigate the effect of chitosan miR-15b-5p nanoparticles on osteogenic differentiation of BMSCs in rats.Methods:Primary rat BMSCs were extracted and osteogenic differentiation was induced to detect the expression changes of miR-15b-5p during osteogenic differentiation.Then,miR-15b-5p was overexpressed in the rat BMSCs and osteogenic differentiation was induced to detect the osteogenic differentiation ability.Furthermore,chitosan/miR-15b-5p nanoparticles with different mass ratios were prepared by ion crosslinking method and their properties were characterized.The osteogenic differentiation ability was detected after transfection of the nanoparticles into BMSCs in vitro and application of animal model in vivo.Among them,the control group without any treatment,using osteogenic differentiation induction solution as the induction group,and the over-expression of miR-15b-5p and osteogenesis induction as the transfection induction group.Results:During osteogenic differentiation of BMSCs in rats,miR-15b-5p expression was up-regulated.After overexpression of miR-15b-5p and induction of osteogenesis,the expression of osteogenic related genes(including RUNX2,OPN and OCN)was higher than that of the induction group and the blank control group.When the mass ratio of chitosan/miR-15b-5p was 20∶1,the encapsulation rate of nanoparticles reached(98.55±0.10)%,the morphology was uniform,the average particle size was 156.6±9.0 nm,and the surface charge was(47.0±0.5)mV.Fluorescence detection showed that chitosan/miR-15b-5p nanoparticles had successfully entered the cytoplasm.The osteogenic differentiation ability of BMSCs increased after application with nanoparticles in vivo and in vitro.Conclusion:MiR-15b-5p can effectively promote osteogenic differentiation of rat BMSCs.Chitosan nanoparticles loaded with miR-15b-5p can promote osteogenic differentiation of BMSCs and bone defect repair.