Objective To investigate the influence of chondrocytes originating from different source on early chondrogenic differentiation of bone marrow mesenchymal stem cells (MSCs) in isolated co-culture system.Methods We applied hanging cell culture system to culture chondrocytes of different origin (osteoarthritis chondrocyte cells,nomal chondrocyte cells,infant chondrocyte cells) and controls.These chondrocytes and MSCs of the same origin were cultured in the common medium in a separated condition,and observed by microscope at 3,6,9,12 day after co culture.Expression levels of aggrecan,collagen type Ⅱ (Col 2),cartilage-specific transcription factor (Sox-9) in MSCs of different origin were determined by Real-time PCR.Results MSCs showed obviously morphological differentiation induced by chondrocytes of different origin at 12 day after coculture as compared with controls.Real-time PCR analysis showed that SOX9 mRNA level was stimulated by 1.7-fold,1.6-fold and 1.2-fold (all P＜0.05) and aggrecan mRNA level was increased by 2.8-fold,2.2-fold and 1.3-fold (all P＜0.05) in infant chondrocytes group,nomal chondrocytes group,osteoarthritis chondrocytes group respectively as compared with controls while COL2 mRNA level had no significant differences among the four groups.Corresponding protein signal level had obvious differences among the four groups,especially in infant chondrocytes as compared with osteoarthritis chondrocytes and nomal chondrocytes.Conclusions Isolated co-culture system may indirectly promote MSCs differentiation to chondrocytes by local micro-environment regulation.Chondrocytes of different origin have different effects on MSCs differentiation,but they could promote MSCs differentiation to chondrocytes.

Objective To construct eukaryotic expression vector of wild type E 4F1 and the mutant deleting amino acid region 32-81, and to detect the interaction between wild type or mutant E 4F1 and p53 and to study the effect of E4F1 on the expression level of p21.Methods Wild type and mutant sequences of E 4F1 were amplified from the mammary library using standard PCR and recombinant PCR .The sequences were cloned into pXJ 40-MYC vector to generate the MYC-E4F1 and MYC-E4F1(Δ32-81) recombinant plasmids that were transfected into 293T cells and identified by Western blotting . FLAG-p53 and MYC-E4F1 or MYC-E4F1(Δ32-81) were co-transfected into 293T cells and immunoprecipitation assay was performed to detect the interaction of wild type or mutant E 4F1 with p53.Wild type and mutant E4F1 expressing vec-tors were co-transfected into osteosarcoma U2OS cells and the expression of p21was detected.Results Recombinant plas-mids of MYC-E4F1 and MYC-E4F1(Δ32-81) were successfully constructed.Both wild type and mutant E4F1 interacted with p53.Deletion of amino acid region 32-81 of E4F1 increased the interaction .The expression level of p21 was in-creased by wild-type E4F1, but not by mutant E4F1.Conclusion The eukaryotic expression vector of wild type E4F1 and its deletion mutant is successfully constructed .Both of them interact with p53.Deletion of amino acid region 32-81 of E4F1 increases the interaction .This study contributes to further studies on the regulation and mechanism of E 4F1 on p53.