| Objective:To construct vectors loading miR-26a based on mesoporous silica nanoparticles(MSNs),and their characterizations and effects on osteogenic differentiation of rat bone marrow mesenchymal stem cells(rBMSCs)were studiedMethod:The MSN_miR-26a@PEI-KALA delivery system was constructed by encapsulating miR-26a in the mesopore of MSNs and modifying polyethylenimine(PEI)and KALA polypeptide on its surface.The morphology,size and Zeta potential of the vectors were investigated by transmission electron microscopy and nanoparticle potential meter.Agarose gel electrophoresis was carried out to evaluate the miRNA protection of the deliveries in vitro.To determine the appropriate transfection concentration and time,we conducted a series of experiments:Cell counting kit-8(CCK-8)assay was used to assess the cell viability of rBMSCs at 12 and 24 h post transfection;Reverse transcriptase polymerase chain reaction(RT-PCR)assay was performed to access the mRNA expression level of miR-26a of rBMSCs at 6,12,and 24 h post transfection.The transfection efficiency of rBMSCs co-incubated with the vectors for 12 h was determined by flow cytometry.The cellular uptake and localization of miR-26a in rBMSCs were observed by confocal laser scanning microscopy.Cells were incubated with MSN@PEI-KALA,MSN_miR-26a@PEI-KALA and MSN_miR-NC@PEI-KALA,respectively,and rBMSCs without treatments were set as control group.ALP staining and activity assay were carried out to detect the osteogenic differentiation in each group after 3 days and 5 days of osteogenic induction.Alizarin red staining and semi-quantitative of calcium nodules assay were carried out to evaluate matrix mineralization level after osteogenic induction for 14 days.The mRNA expression level of Runx2,Opn,Osx and Bmp2 in various groups on the 7th and 14th days after osteogenic induction were accessed by RT-PCR.Western blot was performed to detect the protein levels of OPN,OSX and BMP2 in rBMSCs at 7th and 14th after osteogenic inductionResults:The transmission electron microscopy and nanoparticle potential meter showed that the vectors had good dispersion and positive surface potential.Agarose gel electrophoresis demonstrated that the vectors could protect miR-26a from degradation in vitro.The results of CCK-8 assay and RT-PCT indicated that the appropriate transfection time and concentration were 20 μg/mL and 12 h,respectively,and the transfection efficiency was about 20%～30%.The observation of the laser confocal microscopy showed that the vectors entered the lysosome of the cells through endocytosis and escaped due to the membrane fusion of KALA polypeptide and the "proton sponge" mechanism of PEI,releasing miR-26a into the cytoplasm After 3 days and 5 days of osteogenic induction,a higher ALP activity was observed in the group treated with MSN_miR-26a@PEI-KALA.After 14 days of osteogenic induction,the highest extracellular matrix mineralized level were seen in the group treated with MSN_miR-26a@PEI-KALA.After 7 days and 14 days of osteogenic induction,a higher expression level of related osteogenic genes and proteins were observed in the group treated with MSN_miR-26a@PEI-KALA(P<0.05).Conclusion:The vectors constructed in our study can effectively protect,transport and release mir-26a into the cytoplasm to promote osteogenic differentiation of rBMSCs in vitro,and provide new methods and strategies for the delivery of microRNAs in bone tissue engineering. |
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