Silencing HIF-1α reduces the adhesion and secretion functions of acute leukemia hBMSCs

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.

26S proteasome regulates differentiation of human bone marrow stromal cells into neural-like cells / 中国病理生理杂志

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.

Extracorporeal Shock Wave Promotes Postnatal Human Bone Marrow Stromal Cells Osteogenesis In vivo / 生物化学与生物物理进展

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

Cellular Biocompatibility and Stimulatory Effects of Calcium Metaphosphate on Osteoblastic Differentiation of Human Bone Marrow-derived Stromal Cells / 대한정형외과연구학회지

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.