ABSTRACT
Objective To screen the active components of total flavonoid extracts of Sarcandra glabra to promote megakaryocyte differentiation.Methods(1)A model of megakaryocyte differentiation disorder was established by co-culturing human megakaryocytic leukaemia cells(Dami)with human bone marrow stromal cells(HS-5)as an evaluation system,and the experimental groupings were as follows:the Dami group(Dami),the control group(Dami+HS-5),and the PMA group[Dami+HS-5+5 ng·mL-1 foprolol 12-tetradecanoate 13-acetate(PMA)],and model group[Dami+HS-5+1%rabbit anti-rat platelet serum(APS)+5 ng·mL-1 PMA]were cultured for 48 hours.The expressions of megakaryocyte differentiation and maturation surface marker molecules,CD41a and CD61 were detected by flow cytometry.(2)Forty-nine SD male rats were randomly divided into blank plasma group,15-minute group,30-minute group,60-minute group,90-minute group,120-minute group,and 240-minute group,with 7 rats in each group.The rats in each administration group were gavaged with 1.26 g·kg-1 of total flavonoids extracts of Sarcandra glabra,and blood was collected at six set time points(15,30,60,90,120,240 minutes)for the preparation of time-dependent serum-containing plasma of total flavonoids extracts of Sarcandra glabra.(3)Ultra-high performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry(UHPLC-Q-TOF/MS)was used to analyze the plasma of the time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra,and the peak area was used to construct a matrix(X-matrix)of the amount of chemical composition change over time in the time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra.The collected time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra at six different time points was used to intervene in the model of megakaryocyte differentiation and maturation disorder,and the expression of cell surface molecules CD41a and CD61 was detected by flow cytometry to construct the matrix of effect of time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra(Y-matrix).(4)After the data of X and Y matrices were standardized,partial least squares(PLS)was used to calculate and analyze the quantitative and qualitative effect relationship,and variable importance for projection(VIP)>1 was used as the threshold to screen the effect components related to the changes of cell surface molecules CD41a and CD61,and chemical composition identification,as the potential effector components in the total flavonoid extracts of Sarcandra glabra were used to promote the differentiation of megakaryocytes,and finally the regression evaluation system was used to verify the efficacy of its medicinal effect.Results(1)Compared with the Dami group,the expression level of CD41a on the surface of Dami cells in the control group was significantly increased(P＜0.05).Compared with the control group,the expression levels of CD41a and CD61 on the surface of Dami cells in the PMA group were significantly increased(P＜0.01).Compared with the PMA group,the expression levels of CD41a and CD61 on the surface of Dami cells in the model group were significantly reduced(P＜0.01).(2)Compared with the blank plasma group,the expression levels of the molecules CD41a and CD61 on the surface of Dami cells at each time point of 15,30,60,90,120,and 240 minutes were significantly increased(P＜0.01),and the expression levels of CD41a and CD61 were both highest in the 30-minute group.The potential effective components with VIP value greater than 1 were screened out in the positive and negative ion mode,and 540.3638@12.25 and 559.2991@11.53 were selected for pharmacodynamic verification.559.2991@11.53 was identified as daucosterol(Dau),540.3638@12.25 was identified as rosmarinic acid 4-O-β-D-glucoside(Ros).After Ros and Dau intervened in the megakaryocyte differentiation and maturation disorder model respectively,the expression levels of CD41a and CD61 on the surface of Dami cells in the low-,medium-and high-dose groups(40,60 and 80 μg·mL-1)of Ros and Dau were significantly increased compared with the model group(P＜0.05,P＜0.01).Conclusion Ros and Dau may be the active components of the total flavonoids extracts of Sarcandra glabra to promote the differentiation of megakaryocytes.
ABSTRACT
Hypoxia inducible factor-1α (HIF-1α) is an important transcription factor, which plays a critical role in the formation of solid tumor and its microenviroment. The objective of the present study was to evaluate the expression and function of HIF-1α in human leukemia bone marrow stromal cells (BMSCs) and to identify the downstream targets of HIF-1α. HIF-1α expression was detected at both the RNA and protein levels using real-time PCR and immunohistochemistry, respectively. Vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) were detected in stromal cells by enzyme-linked immunosorbent assay. HIF-1α was blocked by constructing the lentiviral RNAi vector system and infecting the BMSCs. The Jurkat cell/BMSC co-cultured system was constructed by putting the two cells into the same suitable cultured media and conditions. Cell adhesion and secretion functions of stromal cells were evaluated after transfection with the lentiviral RNAi vector of HIF-1α. Increased HIF-1α mRNA and protein was detected in the nucleus of the acute myeloblastic and acute lymphoblastic leukemia compared with normal BMSCs. The lentiviral RANi vector for HIF-1α was successfully constructed and was applied to block the expression of HIF-1α. When HIF-1α of BMSCs was blocked, the expression of VEGF and SDF-1 secreted by stromal cells were decreased. When HIF-1α was blocked, the co-cultured Jurkat cell’s adhesion and migration functions were also decreased. Taken together, these results suggest that HIF-1α acts as an important transcription factor and can significantly affect the secretion and adhesion functions of leukemia BMSCs.
Subject(s)
Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Leukemia, T-Cell/metabolism , Mesenchymal Stem Cells/metabolism , Cell Adhesion , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Jurkat Cells , Real-Time Polymerase Chain ReactionABSTRACT
AIM: To investigate the process of human bone marrow stromal cells (hBMSCs) differentiation into neural-like cells and to determine the role of 26S proteasome in neuronal differentiation. METHODS: Purified hBMSCs were treated with β-mercaptoethanol (β-ME) for 1 day and retinoic acid (RA) for 3 days, followed by growth factor (10 μg/L bFGF or 20 μg/L NGF) for another 3 days. Immunofluorescence was performed to detect the expression of nestin (a neural precursor cells marker), Tuj1 (a premature neuronal marker), and neurofilament (NF, a mature neuronal marker) at all stages of induced differentiation. Immunostaining and RT-PCR were used to analyze the expression of 26S proteasome during neuronal differentiation of hBMSCs. To further confirm the role of 26S proteasome in hBMSCs differentiation, cells were treated with β-ME/RA and then followed by protesome inhibitor MG132 and growth factor. Immunostaining was performed to detect NF-positive cells. RESULTS: Quantification results showed that the untreated cells were almost never positive for nestin, Tuj1 and NF. After treated with β-ME/RA, the numbers of nestin-positive cells (34.41%±1.27%) and Tuj1-positive cells (27.79%±1.27%) were increased. Notably, the numbers of NF-positive cells were significantly increased to 56.72%±2.4% after induction with β-ME/RA/GF. Immunofluorescence analysis showed that undifferentiated hBMSCs cells were weakly stained by antibody against 26S proteasome, but the numbers of cells with high-intensity of 26S proteasome were increased after treated with β-ME/RA. The RT-PCR result of 26S proteasome further confirmed that the mRNA level of the cells differentiated by β-ME/RA (1.33), as well as by β-ME/RA/GF (1.77), was significantly increased compared to the undifferentiated cells. Moreover, hBMSCs incubated with protesome inhibitor MG132 significantly decreased the numbers of NF-positive cells (37.59%±1.52%). CONCLUSION: After induction with β-ME/RA/GF, hBMSCs can be differentiated into neural-like cells, which is concomitant with the increase in 26S proteasome expression. Inhibitor of 26S protesome prevents hBMSCs differentiation, suggesting that 26S proteasome may be involved in the differentiation of hBMSCs into neural-like cells.
ABSTRACT
It is known that extracorporeal shock wave (SW) may promote healing of fracture. A previous study reported that SW promoted human bone marrow stromal cells (hMSCs) towards osteoblasts in vitro. To study the osteogenesis ability of hMSCs treated by shock wave in porous hydroxyapatite (HA) in vivo, primary hMSCs of SW group and control group were cultured in the porous HA for 2 weeks and then implanted into subcutaneous sites of nude mouse. These implants were harvested and prepared for the biochemical analysis of alkaline phosphatase activity by AKP kit, histological analysis of decalcified and undecalcified sections and morphology by scan electric microscope (SEM), as well as osteocalcin mRNA expression by RT- PCR 4 weeks and 8 weeks after implantation. It showed that cells of SW and control group almost covered the rough surface of HA before implantation and the extracelluar matrix of SW group was abundant by SEM photomicrograph . The histological analysis and SEM photomicrograph showed active bone formation 4 weeks and 8 weeks after implantation, as well as tetracycline labeling under fluoroscopy analysis in SW group. Alkaline phosphatase in supernatants of the implants detected 4 weeks and 8 weeks after implantation in SW group was higher than in control group (P
ABSTRACT
PURPOSE: The in vitro biocompatibility of Calcium Metaphosphate (CMP) with human bone marrow stromal cells (HBMSCs) and its effect on osteoblastic differentiation have been evaluated. MATERIALS AND METHODS: The effects of CMP on the HBMSCs undergoing osteoblastic differentiation were evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Morphologies of the HBMSCs were examined using scanning electron microscopy and confocal laser scanning microscopy. Osteoblastic differentiation of the HBMSCs was analyzed by alkaline phosphatase (ALP) staining and RTPCR. RESULTS: The CMP powder and disk did not exert cytotoxic effect on the HBMSCs. In addition, the HBMSCs were adhered on the surface of CMP disk as successfully as on the culture plate or HA disk and displayed similar actin arrangement and cellular phenotypes. Furthermore, the HBMSCs grown on three different matrices were able to support osteoblastic differentiation of the HBMSCs as accessed by ALP staining. However, the CMP disk compared to the HA disk has a better ability to induce expression of osteoblast-related genes such as ALP, osteopontin (OPN) and osteoprotegerin (OPG). CONCLUSION: The results demonstrate that, in addition to biocompatibility of the CMP with the HBMSCs, the CMP has an ability to stimulate osteoblastic differentiation of the HBMSCs in vitro.