Effects of Magnesium Ions on the Calcification of Vascular Smooth Muscle Cells / 天津医药

Objective To explore the effects of the different concentrations of magnesium ions on vascular smooth muscle cell (VSMC) calcification in rats. Methods VSMCs were obtained from rat aortic, and were identified by immunocy-tochemistry. VSMCs were then randomly divided into control group, high phosphorus group and magnesium intervention group. VSMCs were cultured with 10%fetal bovine serum in control group. VSMCs were cultured with high phosphorus in high phosphorus group. VSMCs were cultured with different concentrations of magnesium chloride based on the high phos-phorus medium in magnesium intervention group (final concentrations of magnesium ions were 1, 2 and 3 mmol/L). The calci-um content and alkaline phosphatase(ALP)activity were measured after the stimulation for 7 days. The expression of Cbfα1 mRNA was detected by RT-PCR. Results Compared with control group, calcium deposits were found significantly higher in high phosphorus group and magnesium intervention group. The calcified nodules gradually reduced with the increased magnesium ion concentration in the intervention group. The calcium contents were significantly lower in the intervention groups (2 and 3 mmol/L) compared with those of high phosphorus group (P<0.05), but no difference was found between 1 mmol/L magnesium intervention group and high phosphorus group. There were no significant differences in the ALP activity and Cbfα1 mRNA expression between intervention groups (2 and 3 mmol/L) and control group (P<0.05). The ALP activity and the expression of Cbfα1 mRNA were gradually decreased with the increased magnesium ion concentration in the inter-vention group, and which were lower than those of high phosphorus group (P<0.05). Conclusion Magnesium can reduce calcification and osteoblastic transdifferentiation, which may be achieved by reducing the expression of Cbfα1 in VSMCs.

Expression of Cbfα1/p56 subtype in bone marrow mesenchymal stem cells from rat mandible / 中国组织工程研究

BACKGROUND:Unlike the ilium derived from the paraxial mesoderm, the mandible from cranial neural crest has a unique mechanism. Core binding factorα1 (Cbfα1) is a key transcription factor for skeletogenic process. However, the role of Cbfα1/p56 subtype in mandible tissue is yet not clear. OBJECTIVE:To research the expression of Cbfα1/p56 subtype in bone marrow mesenchymal cells from rat mandible in vitro. METHODS:Bone marrow mesenchymal stem cells from rat mandible and ilium were in vitro isolated and purified by primary culture. The characteristics of bone marrow mesenchymal cells were compared through the methods of enzyme linked immunosorbent assay and real-time PCR, including growth curve, alkaline phosphatase activity and relative mRNA expression of Cbfα1 subtypes. RESULTS AND CONCLUSION:Bone marrow mesenchymal cells from rat mandible and ilium were successful y obtained. Bone marrow mesenchymal cells from the mandible proliferated more rapidly, alkaline phosphatase activity of which was higher than iliac cells. The relative mRNA expression of Cbfα1/p56 subtype in bone marrow mesenchymal cells from the mandible was more than that in iliac cells at 6 days of culture (P0.05). The results showed that Cbfα1/p56 is very significant in the early osteogenic differentiation of bone marrow mesenchymal cells from the mandible.

Dynamic expression of Runx2 gene profile during osteogenesis in stem cells from human exfoliated deciduous teeth / 中国组织工程研究

BACKGROUND:Runx2 is considered to the main regulatory factor of osteogenic gene expression and be necessary for osteoblast differentiation, it plays an extremely important role in the osteoblast development, differentiation, regulation, bone calcification formation and bone repair. OBJECTIVE:To observe the biological properties of mesenchymal stem cells from human exfoliated deciduous teeth, explore the osteogenic differentiation potential of deciduous teeth stem cells, and observe the dynamic expression of Runx2 gene at varying time points. METHODS:The stem cells from human exfoliated deciduous teeth were isolated and cultured in vitro. The cellsurface antigen was detected with flow cytometry. The third passage cells were cultured in the adipogenic medium for 4 weeks, and oil red O staining was conducted to test lipid droplets formation. The third passage cells were cultured in the osteogenic medium for 21 days, and mineralized nodules were detected by alizarin red staining. Runx2 mRNA dynamic expression was detected with semi-quantitative RT-PCR at different time points. RESULTS AND CONCLUSION:The stem cells from human exfoliated deciduous teeth were obtained by enzyme digestion and limited dilution methods. Flow cytometry results showed that, CD146 and STRO-1 were expressed to varying degrees. Oil red O staining revealed salmon pink positive particles. Alizarin red staining showed positive expression. RT-PCR results showed that, Runx2 expression was found at day 0, up-regulated from day 0 to day 6, and subsequently dropped with an expression bottom at day 12, after that a second expression peak occurred at day 18, fol owed by a stably regulation. The stem cells from human exfoliated deciduous teeth can be isolated and cultured in vitro, express surface antigen of mesenchymal stem cells, and have the potentials of differentiating into adipocytes and ostetoblasts. Runx2 gene profiles are dynamical y expressed during osteoblastic differentiation. Runx2 express throughout every stage of osteoblastic differentiation. The expression is up-regulated during early and later stages, and down-regulated in metaphase.

The synergistic regulatory effect of Runx2 and MEF transcription factors on osteoblast differentiation markers

PURPOSE: Bone tissues for clinical application can be improved by studies on osteoblast differentiation. Runx2 is known to be an important transcription factor for osteoblast differentiation. However, bone morphogenetic protein (BMP)-2 treatment to stimulate Runx2 is not sufficient to acquire enough bone formation in osteoblasts. Therefore, it is necessary to find other regulatory factors which can improve the transcriptional activity of Runx2. The erythroblast transformation-specific (ETS) transcription factor family is reported to be involved in various aspects of cellular proliferation and differentiation. METHODS: We have noticed that the promoters of osteoblast differentiation markers such as alkaline phosphatase (Alp), osteopontin (Opn), and osteocalcin (Oc) contain Ets binding sequences which are also close to Runx2 binding elements. Luciferase assays were performed to measure the promoter activities of these osteoblast differentiation markers after the transfection of Runx2, myeloid Elf-1-like factor (MEF), and Runxs+MEF. Reverse-transcription polymerase chain reaction was also done to check the mRNA levels of Opn after Runx2 and MEF transfection into rat osteoblast (ROS) cells. RESULTS: We have found that MEF, an Ets transcription factor, increased the transcriptional activities of Alp, Opn, and Oc. The addition of Runx2 resulted in the 2- to 6-fold increase of the activities. This means that these two transcription factors have a synergistic effect on the osteoblast differentiation markers. Furthermore, early introduction of these two Runx2 and MEF factors significantly elevated the expression of the Opn mRNA levels in ROS cells. We also showed that Runx2 and MEF proteins physically interact with each other. CONCLUSIONS: Runx2 interacts with MEF proteins and binds to the promoters of the osteoblast markers such as Opn nearby MEF to increase its transcriptional activity. Our results also imply that osteoblast differentiation and bone formation can be increased by activating MEF to elicit the synergistic effect of Runx2 and MEF.

Role of interleukin 6 in osteogenic transition and calcification of human umbilical artery smooth muscle cells in vitro and the possible cell signal transduction way / 中华肾脏病杂志

Objective To investigate the role of recombinant human interleukin 6 (rhlL-6) in calcification and osteogenic transition of cultured human umbilical artery smooth muscle cells (HUASMC), and the possible cell signal transduction way. Methods HUASMCs were isolated by the explant method. HUASMCs were treated with (treatment groups) or without (control group) rhIL-6. Alizarin Red S stain was applied for calcium deposition in extracellular matrix of control ceils and the cells treated with rhIL-6 50 μg/L at day 12. Calcium concentration in cell layer of control group and treatment group (treated with rhIL-6 10 μg/L and 50 μg/L, respectively) was determined calorimetrically by the o-cresolphthalein complexone method at day 3, 6, 9 and 12, and corrected by total cell proteins. The mRNA expressions of bone-specific alkaline phosphatase (BAP), osteopontin (OPN), bone morphogenetic protein-2 (BMP2) and osteoprotegerin (OPG) were estimated by real-time PCR in 12, 24 and 72 hours. OPN, BMP2 and OPG expressions were assessed by Western blotting and the BAP concentration at the same time was checked by fluorometry method . Electrophoretie mobility shift assays (EMSA) was used to detect the binding activity of transcription factor Cbfα1 with or without inhibitors of p38-MAPK (SB203580) and PKC (DHC) after 6 hours stimulation by rhIL-6 10 μg/L. Results rhIL-6 induced a positive Alizarin Red S stain and a time-dose-dependent increasing of cell layer calcium deposition.Compared with control group, rhIL-6 10 μg/L enhanced gene expression and protein levels of BAP and BMP2 at the early time (12 and 24 hours), and of OPN and OPG at later hours (24 and 72 hours). RhIL-6 still induced an increasing of binding activity of Cbfα1, which could be partially blocked by DHC but not SB203580. Conclusions rhIL-6 induces HUASMCs calcification and osteogenie transition in vitro, which may be one of the mechanism involved in IL-6 associated vascular calcification as observed in clinical studies. The role of IL-6 in HUASMCs may partially achieved through the PKC cell signal transduction way.