Objective:To study the effect of different concentrations of concentrated growth factor (CGF) on the osteogenic potential of adipose-derived mesenchymal stem cells (ADSCs).Methods:Preparation of CGF from rat blood by differential centrifugation method, the levels of endogenous growth factors in CGF were quantified by enzyme-linked immunosorbent assay (ELISA). ADSCs were extracted from the inguinal adipose tissue of male SD rats and cultured in media containing different concentrations (5%, 20%, 50%, 80%) of CGF. ADSCs were identified by the expression of the surface marker CD44 detected by immunofluorescence staining and by the expression of CD29 and CD90 detected by flow cytometry. Cell proliferation effects was evaluated by the cell counting kit-8 (CCK-8) assay. The effect of osteogenic differentiation was detected by alkaline phosphatase (ALP) staining, and the effect of osteogenic calcification was detected by alizarin red staining. Real-time reverse transcription-PCR was used to detect the expression levels of osteogenic genes, and Western blotting was performed to detect the expression of osteogenesis-related proteins.Results:The detected level of bone morphogenetic protein 2 (BMP-2), transforming growth factor-β1 (TGF-β1), and vascular endothelial growth factor-A (VEGF-A) were (33.15±0.72) pg/ml, (95.60±2.45) ng/ml, and (351.58±22.28) pg/ml in CGF, respectively. ADSCs were spindle-shaped and well-extended. Immunofluorescence staining results showed uniform green fluorescence in the cytoplasm. Flow cytometry analysis revealed that the positive rates for CD29 and CD90 were 99.65% and 92.72%, respectively. The low-concentration (5% and 20%) CGF groups showed stable proliferative activity in ADSCs, with absorbance ( A) value reaching 3.90±0.25 and 4.04±0.22 after 7 days of culture, both of them were higher than that of control group, and the differences were statistically significant (both P<0.01). The 5% CGF group showed the most intense ALP staining, with a cumulative A value of ALP (2.299×10 7±7.156×10 6), which was significantly higher than that in the control group (4.426×10 6±1.839×10 5) ( t=?4.95, P<0.01). All CGF groups showed deeper staining compared to the control group, with the 5% CGF group showing a significantly higher cumulative A value of calcium nodule (3.599×10 7±4.094×10 6) compared to that in the control group (1.413×10 7± 1.298×10 6) ( t=?5.46, P<0.01). The relative expression levels of BMP-2, β-catenin, and TGF-β1 mRNA in the 5% CGF group were 50.97±1.75, 1.84±0.53, and 1.86±0.24, respectively, all of which were significantly higher than those in the control group (26.03±1.94, 1.13±0.12, and 1.14±0.16, respectively) (all P<0.01). Similarly, the protein expression levels of BMP-2, β-catenin, and TGF-β1 in the 5% CGF group were 1 174.33±70.02, 1 337.75±152.88, and1 087.70±112.19, respectively, all of which were significantly higher than those in the control group (845.54±61.97, 670.72±70.95, and 740.70±36.51, respectively) (all P<0.01). Conclusions:Different concentrations of CGF promote ADSCs proliferation, and the lower concentrations (5% and 20%) exhibit more stable proliferation rates. Specifically, a low concentration of CGF (5%) can induce ALP activity and calcium nodule formation, and it enhanced the expression of BMP-2, β-catenin, and TGF-1 mRNA and proteins.

This study was purposed to compare the clinical efficacy and adverse reactions of low-dose decitabine combined with CAG regimen (aclarubicin, Ara-C, and G-CSF) and CAG regimen alone in intermediate to high-risk myelodysplastic syndromes (MDS), and evaluate the validity and efficacy of the former regimen as new treatment method of intermediate to high-risk myelodysplastic syndromes. A total of 12 patients with intermediate (IR) to high-risk (HR) MDS treated by low-dose decitabine combined with CAG regimen and 10 patients with IR to HR MDS treated by CAG regimen alone were evaluated after treatment of 1 cycle and at least after 2 cycles. The complete remission (CR) after 1 cycle, overall remission rate (ORR), progression free survival (PFS) and overall survival (OS) between them were analyzed. The results showed that 9 patients treated by low-dose decitabine combined with CAG regimen achieved complete remission after 1 cycle, 2 patients achieved partial remission, 1 patient did not show reaction. The complete remission rate was 75.0% and overall response rate was 91.7%. The median time of disease free survival was 9 months (0-27 months). The median overall survival time was 16 months (3-28 months). 4 patients suffered from pulmonary infection after treatment and then were all cured after treatment with anti-infective therapy. The 5 patients treated by CAG regimen alone achieved complete remission,3 patients achieved partial remission, 2 patients showed non-reaction. The complete remission rate was 50.0% and overall response rate was 80.0%. The median time of disease free survival was 6 months(0-18 months). The median overall survival time was 13 months(3-31 months), 4 patients suffered from pulmonary infection, 1 patient suffered from enteric infection and 1 patient suffered from Escherichia coli septicemia after treatment, all of them becomed better after active treatment. Two groups of patients all had no serious adverse reactions, All patients could tolerate, no severe complication-related death occurred in them. The statistical analysis indicated that the patients treated with low-dose decitabine combined with CAG regimen had longer progression free survival time than those treated with CAG regimen alone, and had longer overall survival time but did not have statistically significant. It is concluded that low-dose decitabine combined with CAG regimen has better clinical efficacy for patients with intermediate to high-risk MDS and did not increase risk for them. It is worth to apply in clinic.
Aclarubicin ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Azacitidine ; administration & dosage ; analogs & derivatives ; Cytarabine ; therapeutic use ; Disease-Free Survival ; Granulocyte Colony-Stimulating Factor ; therapeutic use ; Humans ; Myelodysplastic Syndromes ; drug therapy ; Remission Induction ; Treatment Outcome