ABSTRACT
OBJECTIVE: To investigate the molecular mechanism of long noncoding RNA (lncRNA) H19 that promotes osteogenic differentiation in rat ectomesenchymal stem cells (EMSCs). MATERIALS AND METHODS: EMSCs were isolated from rat fetal facial processes by flow cytometry. Osteogenic markers CD29, CD90, CD44, CD57, Nestin and sox10 were detected by fluorescent immunoassay. ß-catenin and Wnt pathway target genes were detected by Real-time polymerase chain reaction (RT-PCR) and Western blot after the construction of transient interference H19, a stable expression of H19 EMSCs cell line and the induction of osteogenic differentiation of EMSCs cells. EMSCs of H19 overexpression were treated with Wnt/beta-catenin signaling pathway inhibitor Wnt-C59, and the expressions of beta-catenin and osteogenic markers were detected by RT-PCR and Western blot. Furthermore, the mechanism of H19 regulating Wnt/beta-catenin signaling pathway was explored by transfecting miR-22 and miR-141 mimics and luciferase reporter assays. RESULTS: EMSCs were successfully isolated and identified, osteogenic markers CD29, CD90, CD44, CD57, Nestin and sox10 were significantly overexpressed. Osteogenesis-induced solution significantly increased the expression of H19 and osteogenic markers ALP, Runx2, BMP and OCN in EMSCs (p<0.05). Interference with H19 significantly inhibited the expressions of osteogenic markers, beta-catenin and target genes of Wnt/beta-catenin signaling pathway (p<0.05), while upregulation of H19 significantly promoted the expressions of these markers and genes in EMSCs (p<0.05). Wnt-C59 inhibitors treatment inhibited the Wnt/beta-catenin signaling pathway and osteogenic differentiation in EMSCs with H19 overexpression (p<0.05). Furthermore, H19 could block the inhibitory effect of miR-22 and miR-141 on ß-catenin and activate the Wnt/beta-catenin signaling pathway after transfecting miR-22 mimics and miR-141 mimics in EMSCs (p<0.05). CONCLUSIONS: LncRNA H19 can promote the osteogenic differentiation of rat EMSCs by activating Wnt/beta-catenin signal, providing a theoretical basis for the application of EMSCs in tooth tissue engineering regeneration and repair.
Subject(s)
Mesenchymal Stem Cells/cytology , Osteogenesis/physiology , RNA, Long Noncoding/physiology , beta Catenin/physiology , Animals , Cell Differentiation , Cells, Cultured , Female , Genes, myc , MicroRNAs/antagonists & inhibitors , Pregnancy , Rats , Rats, Sprague-Dawley , Wnt Signaling PathwayABSTRACT
The materials with adsorbent properties were produced from urban sewage sludge by two different procedures via microwave irradiation: (1) by one single pyrolysis stage (SC); (2) by chemical activation with ZnCl(2) (SZ). The BET, SEM and FT-IR have been used to evaluate the pore structural parameters and surface chemistry of the adsorbents, respectively. Subsequently they were used for adsorption of Cu(II) from aqueous solutions. The effects of various experimental parameters, such as pH, temperature were investigated in a batch-adsorption technique. The results showed that the adsorption of Cu(II) was maximal at pH 5.0. The kinetic study demonstrated that the adsorption process was followed the second-order kinetic equation. The experimental adsorption isotherm data were well fitted with Langmuir model and the maximum adsorption capacity of Cu(II) were found to be 3.88 and 10.56 mg/g for SC and SZ, respectively, in the solution of pH 5.0. Thermodynamic parameters such as changes in the enthalpy (ΔH(0)), entropy (ΔS(0)) and free energy (ΔG(0)) indicate that Cu(II) adsorption onto SC and SZ is an endothermic and spontaneous process in nature at 15-45°C. These results indicate that the sewage sludge-derived material via microwave induced ZnCl(2) activation is an effective and alternative adsorbent for the removal of Cu(II) from aqueous solution.
Subject(s)
Copper/chemistry , Sewage/chemistry , Adsorption , Chlorides/chemistry , Entropy , Hydrogen-Ion Concentration , Kinetics , Microscopy, Electron, Scanning/methods , Microwaves , Spectroscopy, Fourier Transform Infrared/methods , Temperature , Thermodynamics , Water/chemistry , Water Pollutants, Chemical/analysis , Zinc Compounds/chemistryABSTRACT
Human pro-urokinase (pro-UK) was cloned into plasmid pET32b and fused to the E. coli thioredoxin (trxA). When expressed in E. coli AD494(DE3), the fusion protein Trx-pro-UK accumulated as insoluble inclusion bodies and amounted to 35% of total cellular proteins. When co-expressed with molecular chaperones human protein disulfide isomerase (PDI) and E. coli GroESL, all the expressed products still existed in the form of insoluble inclusion bodies.
