Chondrogenic Differentiation Of Human Bone Marrow Mesenchymal Stem Cells Induced By Recombinant Human Interferon β1a

Bone marrow mesenchymal stem cells(BMSCs)have the ability of self-proliferation and multidirectional differentiation.They can differentiate into chondrocytes under certain conditions,and can be fused with chondrocytes better after transplantation,so they are ideal cells for cartilage tissue engineering.Our laboratory has been engaged in the research on preparation of recombinant human interferon β and tissue engineering for a long time.The recombinant human interferon β(rhIFN-β)was used as a cytokine in the differentiation of stem cells for the first time.It was found that rhIFN-β could significantly promote the differentiation of human bone mesenchymal stem cells(hMSCs)into chondrocytes,but the mechanism was not clear.The aim of this study was to investigate the effect of rhIFN-β on the directional differentiation of hMSCs chondroblasts.Based on the transcriptional analysis,the possible signal pathways of hMSCs chondrogenic differentiation induced by IFN-β1a were investigated and then the possible signal pathway was verified.This study will lay a theoretical foundation for the new application of rhIFN-β in cartilage tissue engineering.(1)the effects of different concentrations of rhIFN-β with 10 ng/mL TGF-β3chondrogenic differentiation medium on chondrogenic differentiation of hMSCs were studied by the method of inducing pellets.The results showed that the content of GAG in the 100ng/mL IFN-β1a group was significantly higher than that in the other groups(P < 0.05),and the formation of chondrocytes could be promoted by adding 100 ng/mL IFN-β1a on the basis of inducing differentiation and culture with 10 ng/mL TGF-β3.The results of aggregation proteoglycan in the cartilage spheres of alisin blue staining also showed that after the staining,the proportion of blue in TGF-β3+ 100 ng/mL IFN-β1a gourp increased.The expression of mRNA and protein of Sox9 and CollagenII in TGF-β3+ 100 ng/mL IFN-β1a gourp also increased significantly,and the expression of Sox9 and CollagenII increased with the prolongation of induction time.These results suggest that 100 ng/mL IFN-β1a combined with10 ng/mL TGF-β3 can promote cartilage differentiation in hMSCs.(2)The possible signal pathway of hMSCs cartilage differentiation induced by IFN-β1a was analyzed by transcriptome sequencing.The hMSCs was induced for 21 days using the TGF-β3 chondrogenic differentiation medium with100 ng/m L IFN-β1a,and TGF-β3chondrogenic differentiation medium was used to induce hMSCs differentiation as control group.Chondrocytes were collected,RNAs were extracted and differentially expressed genes were sequenced.The results showed that compared with the control group,there were 1349up-regulated genes and 323 down regulated genes in the IFN-β1a group,and the differentially expressed genes were analyzed by GO.The most differentially expressed gene functions included cellular process(1054),single-organism process(938),metabolic process(816),and biological regulation(690),regulation of biological process(646)and response to stimulus(619).The results of Pathway analysis showed that the PI3K/AKT signaling pathway,p53 signal pathway,TNF signaling pathway contributed to the role of IFN-β1a on chondrogenic differentiation of hMSCs.Among these signaling pathway the enrichment effect of PI3K/AKT signaling pathway was the most significant.Therefore,we speculated that PI3K/AKT signaling pathway is closely related to IFN-β1a promoting hMSCs chondrogenic differentiation.(3)In order to investigate the expression of PI3K/AKT during the chondrogenic differentiation of hMSCs,hMSCs were divided into Blank control group、TGF-β3 group and TGF-β3+IFN-β1a group and then were induced into cartilage pellets respectively.At day 4、7、14、21,the pellts were collected and RT-PCR and Western blotting were used to detect the relative expression of PI3 K and AKT.The RT-PCR results showed that the mRNA expression levels of PI3 K and AKT were up-regualted by TGF-β3 induced.Fourthermore,the synergetic effect of TGF-β3 and IFN-β1a would up-regulate the mRNA expression levels of PI3 K and AKT significantly.The Western blotting results showed that TGF-β3 aslo could up-regualte the expression of PI3 K protein and with IFN-β1a the expression level increased significantly.However,acrroding to AKT protein,TGF-β3 maybe only could up-regulate AKT(S473)expression,and with IFN-β1a the AKT(T308)expression could be up-regulated.These results showed that the synergetic effect of TGF-β3 and IFN-β1a could up-regulate the gene expression in PI3K/AKT signaling pathway and then promote hMSCs chondrogenic differentiation.(4)hMSCs were divided into TGF-β3 group,TGF-β3+IFN-β1a group and TGF-β3+IFN-β1a+LY294002 group and then were induced into cartilagepellets respectively.During the chondrogenic differentiation,at day 4、7、14、21,the pellts were collected and RT-PCR and Western blotting were used to detect the relative expression of Collagen II 、Sox9、PI3K and AKT.The results showed that LY29004 could down regulate the expression levels of mRNA and protein of PI3K、AKT、Sox9 and Collagen II.We speculated LY29004 inbited the expression of PI3 K and AKT,which was up-regulated by the synergetic effect of TGF-β3 and IFN-β1a.The role of IFN-β1a on promting hMSCs chondrogenic differentiation was suppressed by inhibiting the activity of PI3K/AKT signal pathway.Therefore,the expression of Collagen II and Sox9 decreased.To sum up,in this paper,the research results show that the ball training IFN-β1a can promote the differentiation of hMSCs into cartilage,containing 10 ng/mL TGF-β3differentiation medium to join 100 ng/mL IFN-β1a can more effectively promote hMSCs differentiation into cartilage,and PI3K/AKT signaling pathway may be mediated the IFN-β1a promote hMSCs differentiation into cartilage,for later IFN-β1a laid a solid foundation in cartilage tissue engineering applications.