[Mage-D1 binding to activated p75NTR positively regulates mineralization of rat ectomesenchymal stem cells in vitro].
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Abstract:
To detect the binding of Mage-D1 with activated p75NTR and explore their role in regulating mineralization of ectomesenchymal stem cells (EMSCs).EMSCs were isolated from the tooth germs of embryonic SD rats (19.5 days of gestation) by tissue explant culture and were identified for surface markers using flow cytometry. The cultured cells were divided into blank control group, 100 ng/mL nerve growth factor (NGF) stimulation group, and lentivirus-mediated Mage-D1 interference (SH-Mage-D1) group. Proximity ligation assay was used to detect the binding of Mage-D1 with activated p75NTR in the EMSCs, and the binding strength was compared among the 3 groups. Alizarin red staining and ALP staining were used to observe mineralization of the induced cells. The expressions of ALP, Runx2, OCN, BSP, OPN, Msx1 and Dlx1 at both the mRNA and protein levels were detected using RT-PCR and Western blotting.The isolated EMSCs expressed high levels of cell surface markers CD44, CD90, CD29, CD146, and CD105 with a low expression of CD45. The results of proximity ligation assay showed that the binding of Mage-D1 with activated p75NTR in the cells increased over time, and the binding strength was significantly greater in NFG-treated cells than in the cells in the other two groups (P < 0.05). Alizarin red staining and ALP staining of the induced cells showed that the changes in the mineralization nodules were consistent with those of ALP activity. The cells treated with 100 ng/mL NGF exhibited significantly increased expressions of ALP, Runx2, OCN, BSP, OPN, Col1, Msx1 and Dlx1 as compared with the cells in the other two groups (P < 0.05).Mage-D1 directly binds to activated p75NTR in embryonic rat EMSCs to positively regulate the mineralization of the EMSCs.Keywords:
RUNX2
CD146
ALIZARIN RED
Adult adipose tissue-derived stem cells (ADSCs) were found to hold great promise for use in bone tissue repair and regeneration. The present study aims to improve the osteogenesis of ADSCs by Superparamagnetic Iron Oxide (SPIO), which is widely used in tissue imaging. In this study, adipose-derived stem cells were harvested from 4-week-old male Sprague-Dawley (SD) rats. The proliferation rates of ADSCs labeling with or without SPIO were assessed by using trypan blue assay. The osteogenic capability was examined by employing the ALP activity detection kit. The mineralization of cells was determined by staining with Alizarin red S. Flow cytometry analysis was used to examine the cell apoptosis treated with or without SPIO. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis was utilized to detect the Runx2, Opn, Ocn and ALP genes in the cells. The results indicated that SPIO could promote rat ADSCs proliferation and reduce rat ADSCs apoptosis. Also, SPIO could significantly enhance the ALP and alizarin red staining of ADSCs in -SPIO group and +SPIO group (P < 0.01). Furthermore, we also found that the expression of Runx2, Opn, Ocn and ALP was significantly increased after SPIO treatment compared to the un-treated cells (P < 0.01). In conclusion, SPIO could promote the osteogenic differentiation of rat adipose-derived stem cells, which would also become a great potential therapeutic tool in bone tissue engineering.
RUNX2
ALIZARIN RED
Trypan blue
Osteopontin
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Propidium iodide
Nuclear DNA
SYBR Green I
Bromodeoxyuridine
Cytometry
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Differential centrifugation
CD69
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Cell Sorting
Cytometry
Fluorescent labelling
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Propidium iodide
Fixative
Feulgen stain
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Ex vivo
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Background Dopamine exerts its effects mainly in nervous system through D1, D2 or D3 receptors. There are few reports dealing with the effects of dopamine on leukaemia cells. However, some dopamine agonists or antagonists do show biological effects on some types of leukaemia cells. Here, we report the effects of dopamine on the proliferation, differentiation and apoptosis of K562 leukaemia cells. Methods Proliferation was determined by MTT assay and cell counting both in liquid and semisolid cultures. Differentiation was verified by morphology, benzidine staining and flow cytometry. Apoptosis was checked by Hoechst 33258 staining and flow cytometry. The two groups were untreated group and treated group (dopamine 10−9 mol/L-10−4 mol/L). Results In liquid culture, MTT assay and colony assay, dopamine inhibited proliferation of K562 cells. Inhibition rate was 29.28% at 10−6 mol/L and 36.10% at 10−5 mol/L after culture for 5 days in MTT assay. In benzidine staining and CD71 expression, dopamine induced K562 cells toward erythroid differentiation by increased 155% at 10−6 mol/L and by 171% at 10−5 mol/L after culture for 5 days in benzidine staining. In Hoechst 33258 staining and flow cytometry, dopamine induced K562 cells toward apoptosis. The sub G1 peak stained by PI was 14.23% at 10−4 mol/L dopamine after culture for 3 days compared with the control (0.81%) in flow cytometry. Conclusion Dopamine inhibites proliferation and induces both differentiation and apoptosis of K562 leukaemia cells. Chin Med J 2007;120(11):970–974
MTT assay
K562 cells
Benzidine
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Agarose gel electrophoresis
Fragmentation
Growth inhibition
MTT assay
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