A Study On Expression Of Gene Cx3cl1 Under Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) are considered to bemultipotent stem cells that are easily induced to differentiate into functionalosteoblasts both in vitro and in vivo. Easily obtained and expanded, BMSCs havebeen supposed to be the most hopeful cell source for the application of tissueengineering and regenerative medicine. Although the inductive system forosteogenic differentiation of BMSCs is well established, the molecular mechanismunderline the differentiation of BMSCs is not so clear.According to our previous research on the gene expression pattern ofdifferentiated BMSCs applying micro-array chip, the expression of cx3cl1 genes isclosely related to osteogenic differentiation of BMSCs. In this study, the real-timePCR experiment are performed to analysis cx3cl1 gene expression pattern in stageof BMSCs differentiation in vitro in order to further reveal the potential functionaleffect ofcx3cl1 gene.BMSCs were isolated and purified from rat tibia, applying density gradientcentrifugation combined with different adherent culture. BMSCs were thenexpanded and induced in osteogenic medium. Several assays were performed todetermine the growth and differentiated properties of BMSCs: MTT for growthcurve, qualitative and quantitative analysis of alkaline phosphatase activity, typeâ…  collagen staining and Von Kossa's staining for mineralized nodules. RT-PCR wasthen used to detect the expression of cx3cl1 in BMSCs at different stages ofosteogenic differentiation. Finally Real time PCR was applied to examine thequantitative change of cx3cl1 expression in at different stages of osteogenicdifferentiation. Our results indicated that rat BMSCs obtain from density gradientcentrifugation and different adherent culture, are easily induced to differentiate intofunctional osteobalsts in osteogenic medium with enhancement of alkalinephosphatase activity, formation of mineralized nodules and deposition ofextracellular matrix. Further more, the expression of cx3cl1 in BMSCs increasedafter osteogenic induction, with a peak at 3w. Gene expression levels betweeninduced cells and un-induced cells are significantly different.Taken together, our results lead to a conclusion that expression of cx3cl1increases with osteogenic differentiation of BMSCs, and this gene may playimportant roles in osteogenic differentiation of BMSCs and their mineralization.