| Objective: The purpose of this study is to detect the changes of osteogenic capacity and senescence of human bone marrow mesenchymal stem cells(BMSCs)during ageing;and to explore the differential gene expression profiles of human bone marrow mesenchymal stem cells in different age groups by gene chip technology in order to provide a theoretical basis for bone marrow mesenchymal stem cells as seed cells in tissue regeneration.Method: 1.Collection,isolation,culture and identification of BMSCs in different age groups According to age,BMSCs were divided into fetal-BMSC group(20-24 weeks,n=6),young-BMSC group(16-30 years old,n=6),old-BMSC group(over 60 years old,n=6).Flow cytometry was used to detect the surface markers of mesenchymal stem cells.The osteogenic and adipogenic differentiation ability of the isolated cells was identified by alizarin red staining and oil red O staining respectively.2.Detection of osteogenic differentiation of BMSCs in different age groups Bone marrow mesenchymal stem cells of different ages were cultured and differentiated into osteoblasts for 2 weeks.The osteogenic differentiation ability of mesenchymal stem cells was identified by alizarin red staining.The expression level of key genes in osteogenesis was verified by Real-time PCR.3.To detect the senescence degree of BMSCs in different age groups.The senescence degree of BMSCs of different age groups was detected by cell aging beta-galactosidase staining.Real-time PCR was used to detect the expression of aging-related gene P21?P16.The cell cycle of human bone marrow mesenchymal stem cells was detected by flow cytometry.4.Related gene profiles of BMSCs in different age groups Microarray samples of different age groups were prepared according to the requirements.The samples were stored at-80?.Sequencing and bioinformatics analysis were carried out.Results: 1.Flow cytometry showed that CD90 and CD44 were positive,while CD45 and CD19 were negative.After 2 weeks of osteogenesis and adipogenesis induction,alizarin red staining and oil red O staining were positive.2.Bone marrow mesenchymal stem cells of different age groups were differentiated into osteoblasts.Alizarin red staining showed that the calcium nodule formation ability of BMSCs in fetal group was better than that in other two groups.Calcium nodules were the lowest in the old-BMSC group(P?0.01).Real-time PCR results showed that the expression of osteogenesis-related genes RUNX2,COL1A1 and ALP decreased with age(P?0.01).3.The positive rate of ?-galactosidase staining cells increased with age.Real-time PCR showed that the expression of aging-related marker genes P21 and P16 in the old group was significantly higher than that in the young group and the fetal group.The lowest expression was found in the fetal group(P?0.01).The number of human bone marrow mesenchymal stem cells in G1 phase elevated with age(P?0.01).4.To screen differential expression gene,the results of microarray showed that there were 601 up-regulated genes and 508 down-regulated genes in the old group compared with the fetal group,577 up-regulated genes and 550 down-regulated genes in the young group compared with the fetal group,and only 15 up-regulated genes and 7 down-regulated genes in the old group compared with the young group(Fold change?2,P? 0.05).Trend analysis of common differential expression genes among the three groups showed that there were 28 genes(Fold change ? 1.2,P ? 0.05),three of which increased with age;the expression of the young group and the old group decreased compared with the fetal group,but the expression of the old group increased by 9 genes compared with the young group,and the expression of the decreased genes with age increased by 16 genes.Five of the 28 genes had a multiple of gene difference ? 2 and a P value ? 0.05,including TACSTD2,PRKCQ-AS1,WFDC21 P,NAP1L2,COL4A5.Conclusion: 1.Isolated cells were human bone marrow mesenchymal stem cells.Bone formation ability of BMSCs in fetal group was better than that in other two groups.2.The senescence degree of human bone marrow mesenchymal stem cells increases with age.3.Compared with the fetal group,the gene expression of the young group and the old group was significantly different.There was little difference between the young group and the old group.The trend analysis of differential expression genes in the three groups showed that there were 28 genes with differentially expressed multiple ? 1.2 and with significant difference.Five of the 28 genes had a multiple of gene difference ? 2. |
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