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Aim To investigate the injury of 5-fluorouracil(5-FU)to perivascular hematopoietic niche via isolating mouse bone marrow perivascular mesenchymal progenitor cells in vitro and its related mechanism. Methods The perivascular mesenchymal progenitor cells were isolated from femurs and tibias of C57BL/6J mice with type Ⅱ collagenase and cultured in vitro. Agarose gel electrophoresis was used to detect specific niche genes expression. The viable cells were counted by Trypan blue; the cellular proliferation was detected by CCK-8; the apoptosis was detected by Annexin V/PI double staining, and the cell senescence was detected by β-galactosidase staining. The levels of malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by enzymatic assay. Osteogenic and adipogenic differentiation potential of cells were detected by osteogenic and adipogenic differentiation experiment and osteogenic related genes qRT-PCR assay. The mRNA expression of hematopoietic growth factors was detected by qRT-PCR. Hematopoietic cells were co-cultured with perivascular mesenchymal progenitor cells, and the adhesion molecules and signal molecules between stromal cells and hematopoietic cells were detected, also hematopoietic cell activity, redox indicators and β-galactosidase specific cell senescence were detected. Results 5-FU caused simultaneous apoptosis and senescence of perivascular mesenchymal progenitor cells, inhibited cell proliferation, induced oxidative stress, led to osteogenic/adipogenic differentiation imbalance, and down-regulated the transcription of hematopoietic factors SCF, CXCL12, and G-CSF. For the interaction between stromal cells and hematopoietic cells, the binding effects of VLA-4/VCAM-1, ICAM-1/LFA1 were weakened and TPO/MPL and ANG-1/Tie-2 signals were impaired, leading to oxidative stress of hematopoietic cells and cell senescence. Conclusions 5-FU induces oxidative damage of perivascular mesenchymal progenitor cells and indirectly induces premature senescence of hematopoietic cells.
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Objective To investigate the effect of adenovirus-bone morphogenic protein 9 ( Ad-BMP9 ) on osteogenic differentiation of immortalized calvarial mesenchymal progenitor cells ( iCALs ) .Methods iCALs were infected with adenoviral vectors encoding BMP-9 or green fluorescent protein ( GFP) and the early osteogenic differentiation was assessed by detecting alkaline phosphatase (ALP) activity after being cultured for 3, 5 and 7 days.14 days after infection, alizarin red S staining was performed to study the formation of osteogenic calcium nodules .The expression of osteogenic marker genes Runx2 and OCN was assessed by quantitative real-time ( RT )-PCR and Western blotting .Results Significant increases in ALP activity and in the expressions of Runx 2 and OCN were detected in BMP-9 treated iCALs compared with GFP-treated cells(P<0.05).Meanwhile, alizarin red S staining showed that more mineralized nodules were found in the BMP-9 induced group .Conclusion BMP-9 can promote the osteogenic differentiation of iCALs .
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Objective To observe the growth and proliferation capabilities of MPCs in primary OA articular cartilage and their differen-tiation properties into chondrocytes by applying related genes SOX6 and SOX9, so as to provide theoretical evidence in preventing and curing primary OA. Methods SOX6 and SOX9 genes were respectively ligated into adenovirus shuttle plasmids pAdTrack-CMV-SOX6 and pAdTrack-CMV-SOX9, then the recombinant plasmids were used to infect MPCs derived from primary OA articular cartilage. TB and the ex-pressions of collagen type Ⅱ protein and mRNA in differentiated MPCs were compared between the infected group and the uninfected group. Results Either SOX6 gene or SOX9 gene could stably infect MPCs from primary OA cartilage. TB and collagen typeⅡwere strongly posi-tive in the SOX6-infected or SOX9-infected MPCs, while they were weekly positive in the uninfected MPCs. Collagen typeⅡmRNA expres-sion in SOX6-infected MPCs derived from primary OA cartilage was 3. 8 times of that in uninfected cells (P<0. 01), and that in SOX9-in-fected MPCs was 5. 15 times of that in the uninfected cells (P<0. 01). Conclusion The stable transfection of SOX6 and SOX9 genes into MPCs derived from primary OA cartilage could significantly promote chondrogenic differentiation of MPCs. There must be feasible methods of gene technology to promote cell proliferation and differentiation of MPCs for repairing articular cartilage injury.
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Objective To observe the regulatory effect of bFGF and TGF-β1 for the proliferation of mesenchymal progenitor cells ( MPCs) derived from primary osteoarthritis cartilage, and to provide theoretical evidence in preventing and curing primary OA. Methods Different concentrations of bFGF and TGF-β1 ( alone or combined) were used to treat primary OA cartilage and their effects on proliferation of MPCs were tested by MTT method. Results Either bFGF (10. 0~50. 0 ng/mL) or TGF-β1 (0. 1 ~1. 0 ng/mL) alone can significantly promote the proliferation of MPCs derived from primary OA cartilage (P0. 05). The combination of 10. 0 ng/mL bFGF and 1. 0 ng/mL TGF-β1 significantly increased the prolifer-ation of MPCs from primary OA (P<0. 05). Conclusion Both bFGF and TGF-β11 play important roles in the proliferation of MPCs in primary OA cartilage,and they can increase the proliferation in different degree with different concentrations. There must be feasible methods of gene technology to promote cell proliferation and differentiation of MPCs for repairing articular car-tilage injury.
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Objective:To culture mesenchymal progenitor cells(MPC)from compact bone fragments in C57 mice in vitro and to study the feasibility of inducing directed differentiation of MPC into neuron-like cells in vitro. Methods:Bones of hind limbs of C57 mice were sheared into bone fragments and digested by collagenase type Ⅱ. Then,MPC were cultured in vitro and analyzed by flow cytometry for identification of its immunology phenotype. MPC of P3 in good growth were induced directionally by the supernatant cultured with primary neuron,and then detected the expression of neuronal specific markers neuron specific enolase(NSE)and neurofilament protein(NF)by immunocytochemical staining. Results:The primary MPC were cultured successfully and they grewwell after passage. Compared with that of the control group,the positive rate of CD29 and CD44 of MPC of antibody group had significant difference (P