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BACKGROUND: The source of bone marrow mesenchymal stem cells (BMSCs) is limited, and the cellular morphology,proliferation and multi-directional differentiation capacities can vary during serial passages in BMSCs in vitro.OBJECTIVE: To study the effects of fibroblast growth factor (FGF) on cellular morphology, proliferation and differentiation of serially passaged BMSCs.METHODS: (1) BMSCs were isolated from Sprague-Dawley rats and cultured. These cells were passaged six times in vitro, and the cellular morphology was observed and photographed. (2) BMSCs at passage 6 were seeded into 96-well plates and randomly divided into control group and FGF treatment group. The proliferation of cells in both groups was detected with cell counting kit-8 kit at days 1, 2, 3, 4, 5, 6, 7 after culture. (3) BMSCs at passage 6 were seeded into 6-well plates and randomly divided into control group and FGF treatment group. After 7 days treatment with growth medium or growth medium containing FGF, the cellular morphology was observed and photographed. And then the cells of both groups were treated with osteogenic induction medium, adipogenic induction medium and chondrogenic induction medium for the next 7 days. The osteogenic, adipogenic and chondrogenic related genes (RUNX2, ALP, OCN;PPARγ2, AP2, ADIPOQ; SOX9, collagen II, aggrecan) were detected with real-time PCR. The protein expressions of RUNX2, PPARγ2, SOX9 were detected with western blot assay. (4) BMSCs at passage 6 were seeded into 6-well plates and randomly divided into control group and FGF treatment group. After 7 days treatment with growth medium or growth medium containing FGF, the cells were cultured with osteogenic induction medium, adipogenic induction medium and chondrogenic induction medium for the next 14 days. Then, alizarin red S staining, oil red O staining and alcian blue staining were performed.RESULTS AND CONCLUSION: (1) After in vitro passage for six times, the cellular morphology changed obviously, and FGF treatment recovered the characteristics of primary cells. (2) Compared with the control group, the cell proliferation in the FGF treatment group was significantly increased (P < 0.05). (3) Compared with the control group, the expression of osteogenic, adipogenic and chondrogenic related genes (RUNX2, ALP, OCN; PPARγ2, AP2, ADIPOQ; SOX9, collagen II, aggrecan) was increased significantly in the FGF treatment group (P < 0.05). The protein expressions of RUNX2,PPARγ2, SOX9 were also higher in the FGF treatment group than the control group (P < 0.05). (4) Compared with the control group, the number of extracellular calcium nodules, the number of intracellular lipid droplets, and the expression of acid acidic mucopolysaccharide were significantly increased after FGF pretreatment. To conclude, FGF pretreatment can preserve the stemness of BMSCs serially passaged in vitro.
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Objective To explore the effects of electromagnetic fields (EMFs) on osteogenesis during co-culture of bone mesenchymal stem cells (BMSCs) with osteoblasts in rats.Methods BMSCs and osteoblasts were isolated from Sprague-Dawley rats and cultured.Sub-cultured osteoblasts and BMSCs were seeded in transwell cell-culture-chamber polyester inserts to establish the co-culture system.The co-cultures were then randomly divided into a normal co-culture group and a group exposed to an EMF.Single-cultured BMSCs and osteoblasts were set as a single culture group.The EMF group was exposed to an EMF for 4 hours per day.On the 14th day,cell culture plates or inserts were randomly selected for total RNA extraction and measurement of the mRNA expression levels of Runt-related transcription factor 2,transcription factor 7,alkaline phosphatase,collagen type Ⅰ,bone morphogenetic protein 2 (BMP-2) and bone gamma-carboxyglutamate protein (Osteocalcin gene,OC gene) using real-time PCR assays.Cell culture dishes or inserts were also randomly chosen for Alizarin red staining to detect mineralized nodules.Results The level of osteogenic gene expression in single-cultured BMSCs and osteoblasts was low,while it was much higher in the co-culture group.The level of gene expression in the EMF-exposed and co-cultured group was even higher.Alizarin red staining also showed that calcium mineralized modules had increased in the stimulated,co-cultured system compared with the unstimulated,co-cultured cells.Conclusion EMF exposure can promote osteogenic differentiation of BMSCs and osteoblasts when they are co-cultured.BMP2-mediated cellular interaction might play an important role in osteogenic differentiation induced by EMF exposure.
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OBJECTIVE@#To analyze cerebral hemodynamic changes by means of transcranial Doppler (TCD) and generally to explore the clinical application of this method in hyperlipemia patients.@*METHODS@#Cerebral hemodynamics were detected by TCD in 63 patients with hyperlipidemia and compared with the hemodynamics of 64 health people.@*RESULTS@#No statistically significant changes were found in the cerebral artery blood flow velocity and pulsatility index between the hyperlipidemic and control groups (P>0.05). Spectral shape, however, was abnormal in 52 patients in the hyperlipemia group (82.54%), which was statistically different (P<0.005) from controls. These abnormalities were classed as follows: 22 patients had abnormal spectra of the vertebrobasilar system, 2 patients had abnormal spectrum of the internal carotid arterial system, and 28 patients had abnormal spectra of both systems. The incidence of the abnormal spectra in vertebrobasilar system was significantly higher than the internal carotidartery (P<0.005).@*CONCLUSION@#TCD examination can reveal abnormal spectral shape in the cerebralartery and vertebrobasilar arterial systems in hyperlipidemia patients, and thus has some clinical value in determining changes in the brain of patients with high cholesterol levels and atherosclerosis.
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Adult , Female , Humans , Male , Middle Aged , Cerebrovascular Circulation , Physiology , Hemodynamics , Physiology , Hyperlipidemias , Diagnostic Imaging , Ultrasonography, Doppler, TranscranialABSTRACT
OBJECTIVE@#To investigate the diagnostic value of transcranial Doppler ultrasound in early stage cerebral arteriosclerosis.@*METHODS@#We selected 50 patients with early cerebral arteriosclerosis as the disease group. At the same time we selected another 50 patients as a control group with no significant symptoms in the nervous system. By 2 MHz pulse Doppler probe through double-temporal windows and pillow windows Basilar artery (BA), the bilateral middle cerebral artery (MCA) was detected. In the TCD spectrum, we selected the spectrum of a single-family cardiac cycle, identified the starting point (ts), pulse-incisure point (ti), end point (te), and the peak velocity (tp), measured the time of the spectrum starting point to the peak velocity (Tp) and calculated the time required for the peak velocity in the share of ventricular systolic (Tp/Ti), the time required for the peak velocity in the share of the whole cardiac cycle (Tp/T). Tp, Tp/Ti and Tp/T were respectively named as time to peak velocity (TPV), peak-time index-1 (PTI-1) and peak-time index-2 (PTI-2). All data were analyzed by SPSS13.0.@*RESULTS@#There was no significant difference in blood vessel velocity, PI, RI and S/D of BA and RMCA (P>0.05) between the control group and the disease group. Compared with the control group, TPV of the BA, LMCA and RMCA significantly extended, PTI-1 and PTI-2 of BA, LMCA and RMCA increased significantly in the disease group (P0.05).@*CONCLUSION@#TPV, PTI-1 and PTI-2 are sensitive indicators of early stage cerebral arteriosclerosis.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Basilar Artery , Diagnostic Imaging , Blood Flow Velocity , Physiology , Case-Control Studies , Intracranial Arteriosclerosis , Diagnostic Imaging , Middle Cerebral Artery , Diagnostic Imaging , Ultrasonography, Doppler, TranscranialABSTRACT
Objective To explore in vitro the best time window for using sinusoidal electromagnetic fields to promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).Methods BMSCs were isolated and cultured from 4-week-old Sprague-Dawley rats (male and female,80-120g).The BMSCs (from passage 3) were exposed 0,1,4 or 8h/d for 7d,14,or 28d,respectively,to 15Hz sinusoidal electromagnetic fields with a maximum amplitude of lmT.Those exposed 0h/d served as the control.The relative expressions of runt related gene-2 (RUNX2),bone sialoprotein (BSP) and osteopontin (OPN) were determined using real-time,quantitative reverse transcription-polymerase chain reactions (RT-PCRs).The level of RUNX2 protein was determined by Western blotting after 14d.Alizarin red staining was used to compare calcium distribution in each group.Results Obvious promotion of differentiation to osteoblasts was observed after 7 days of exposure to the15 Hz sinusoidal electromagnetic fields,most obviously manifested by an outstanding increase of the early osteogenic index RUNX2 in those exposed 4h/d.After 14 days of intervention,the 1h/d exposure showed to be most effective,especially in inducing the changes of the late osteogenic index OPN.The trends of changes in RUNX2 protein were similar in all groups.After stimulating 1h/d for 14 and 28days,calcium deposition increased to the greatest extent.Conclusions Exposure to sinusoidal electromagnetic fields induces osteogenic differentiation to osteoblasts in rat BMSCs in vitro.There is an apparent window effect.The best results are observed with more days of exposure and shorter exposure time (1h) every day.
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Objective To investigate the biological effect of 50 Hz sinusoidal electromagnetic fields at 1 mT on the proliferation of the human osteosarcoma cell line MG-63.Methods Osteosarcrma MG-63 cells were divided into control and experimental groups.The control group was incubated without an electromagnetic field; the experimental group was incubated in a 50 Hz,1.0 mT sinusoidal electromagnetic field.On the 2nd,4th and 6th day,their proliferation was determined using a cell counting kit-8(CCK-8)assay.Variations in the cell cycle were detected with flow cytometry(FCM).Semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR)was used to measure cyclin B1 and cyclin D1 mRNA.Results Compared with the control group,proliferation of the experimental group cells was reduced significantly.The percentage of cells at G0-G1 phase increased,and the mRNA expression of cyclin B1 and cyclin D1 was significantly reduced.Conclusions A 50 Hz sinusoidal electromagnetic field at 1.0 mT can inhibit the proliferation of osteosarcoma cell line MG-63 significantly.