| Mesenchymal stem cell(MSC)is an adult stem cell derived from the mesoderm,which can differentiate to form different tissues such as bone,fat,and nerve.It is the ideal seed cells for tissue engineering and play an important role in tissue repair.However,the mechanism of its multipotential differentiation restrain unclear.In our laboratory,we found that chitinase 3-like protein(CHI3L1)is highly expressed in human umbilical cord MSC(h UC-MSC)through transcriptome sequencing analysis,but its regulation of MSC multilineage differentiation have not been reported.Thus,we investigate the effect and mechanism of CHI3L1 in the regulation of multipotential differentiation of h UC-MSC.It will provide theoretical and experimental basis for elucidating the mechanism of multipotential differentiation of MSC.First,hUC-MSC were resuscitated and cultured,the cell surface markers CD14,CD34,CD45,CD73,CD90,CD105 were detected by flow cytometry,and the differentiation ability of adipocytes and osteocytes in vitro was assessed by oil red O staining,alkaline phosphatase(ALP)staining and alizarin red staining,and the m RNA expression levels of key adipogenic and osteogenic transcription factors were examined by real-time fluorescence quantitative PCR(q PCR).Next,hUC-MSC with stable knockdown of CHI3L1(sh-CHI3L1-MSC)and control h UC-MSC(sh-NC-MSC)were constructed using lentiviral vectors.Oil red O staining,ALP staining,alizarin red staining and q PCR were used to explore the effect of CHI3L1 on adipogenic and osteogenic differentiation of h UC-MSC.The results showed that cultured h UC-MSC was highly expressed CD73,CD90,CD105,and low or no expressed CD14,CD34,CD45,and CHI3L1 knockdown did not affect the expression of h UC-MSC surface markers.After osteogenic differentiation,compared with the self-differentiated group,the activity of ALP,the number of mineralized nodules and the expression of ALP and OPN,the key transcription factors of osteogenic differentiation,were increased significantly in the induced group(P<0.001).Adipogenic differentiation assay showed that a large number of red lipid droplets appeared in the induced group after oil red O staining,and the expression of adipogenic transcription factor ADI and PPAR-γ were significantly increased in the induced group by q PCR assay(P<0.001).These results demonstrated that cultured h UC-MSC had the ability of osteogenic and adipogenic differentiation.Further,the multipotential differentiation ability of h UC-MSC with knockdown of CHI3L1 was examined.The results showed that ALP activity was significantly reduced in the sh-CHI3L1-MSC osteogenesis-induced group compared with the sh-NC-MSC group,and alizarin red staining showed a significant reduction in mineralized nodules.QPCR assay showed that the relative expression of m RNA levels of ALP,OPN,and DLX5 were significantly reduced in induced group(P<0.05).These data suggested that the osteogenic differentiation ability of h UC-MSC with knockdown of CHI3L1 was diminished.Meanwhile,the number of lipid droplets was significantly increased in the sh-CHI3L1-MSC adipogenesis-induced group compared with the sh-NC-MSC group,and the relative expression of m RNA levels of ADI and PPAR-γ were both significantly higher(P < 0.001).It was suggested that knockdown of CHI3L1 in h UC-MSC had enhanced adipogenic differentiation ability.The above results indicated that the CHI3L1 gene was involved in the regulation of osteogenic-adipogenic differentiation of h UC-MSC in vitro,and it could promote the osteogenic differentiation of h UC-MSC and inhibit the adipogenic differentiation,suggesting that the CHI3L1 gene plays a role in the adipogenic and osteogenic differentiation of MSC,and could be a new target for the regulation of multipotential differentiation of h UC-MSC. |
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