关键词: Bmi-1基因, p16基因, 骨髓间充质干细胞, 老化

Abstract:

Abstract:Objective To study the changes of Bmi-1 and p16 gene expressions in human fetal bone marrow-derived mesenchymal stem cells(MSCs) with the increasing of passage number,and to clarify  the relationship between Bmi-1,p16 gene expressions and replicative senescence of MSCs. Methods By using the adherence culture method,MSCs were isolated from the femoral bone marrow of three abortuses: 16-week-old,17-week-old and 25-week-old,respectively; and then the MSCs were isolated,cultured and amplified. The expressions of CD13,CD34，CD44，CD45，CD73，CD90，CD105，CD133 and HLA-DR in the fifth generation of MSCs were detected with flow cytometry. After successively subcultured,the fetal bone marrow-derived MSCs were induced to replicative senescence. The proliferation  abilities of MSCs at passage 5(P5),passage 15(P15) and passage 30(P30) were detected with BrdU incorporation assays. The expression levels of Bmi-1 and  p16 gene mRNA in MSCs at P5,P15 and P30 were determined with Real-time PCR. Results The MSCs were isolated from the femoral bone marrow of the three abortuses and then cultured successfully,with positive expressions for CD13,CD44, CD73,CD90,CD105 and almost no expressions for CD34,CD45,CD133 and HLA-DR; With the increasing of passage number,the cell growth rate was  gradually slowed down,and the cells became flat,and the refractivity was decreased. The BrdU incorporation rates of  MSCs between P5 and P15 had no significant difference（P>0.05），but the BrdU incorporation rate of  MSCs at P30 was lower than those of MSCs at P5 and P15（P<0.05）. The expression level of Bmi-1 gene mRNA of MSCs at P5 was higher than those of MSCs at P15 and P30 (P<0.05),and the expression level of Bmi-1 gene mRNA of MSCs at P15 was higher than that of MSCs at P30 (P<0.05). The expression levels of p16 gene mRNA of MSCs between P5 and P15 had no significant difference（P>0.05），but the expression level of p16 gene mRNA of MSCs at P30 was lower than those of MSCs at P5 and P15 (P<0.05). Conclusion MSCs can be successfully isolated from the femoral bone marrow of the human fetus. The replicative senescence of fetal bone marrow-derived MSCs has correlation with the mRNA expression level of Bmi-1,but the signal pathway may not mediate through p16INK4a-Rb pathway .

Key words: Bmi-1 gene, p16 gene, bone marrow-derived mesenchymal stem cells, senescence