| Objective: To investigate the effect of augmenter of liver regeneration(ALR)on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).Methods: The recombinant plasmid pPICZalpha A-rhALR was digested by restriction endonuclease Sac I and electroporated into yeast GS115.High copy-positive transformed yeast colonies were screened on YPD plate containing Zeocin,and rhALR was secreted by methanol induction.The expressed protein was purified by nickel column affinity chromatography and identified by SDS-PAGE gel electrophoresis and Western Blot.CCK-8 reagent was used to detect the activity of rhALR on human hepatocellular carcinoma cell line HepG2.Bone marrow mesenchymal stem cells(BMSCs)were isolated in vitro by cell adherence separation assay,and purified continuously through successive passage.Morphological changes of BMSCs were observed under microscope,and surface antigen markers of BMSCs were detected by flow cytometry.CCK-8 assay was used to detect ALR(1-100ug/mL)group and control group at different concentrations.The effect of ALR on each group after 48 hours was observed.Alkaline phosphatase assay kit was used to detect the changes of alkaline phosphatase activity in ALR group and classical osteogenic induction group 7 days after osteogenic induction.Alizarin red staining was used to detect the formation of calcium nodules in ALR group and classical osteogenic induction group 14 days after induction,and real-time fluorescence quantitative PCR was used to detect the expression of osteogenic related genes Runx2,OCN and OPN after 14 days of osteogenic induction.Results:1.The recombinant plasmid was identified by restriction enzyme digestion and sequencing.rhALR was secreted and expressed in the supernatant.After nickel column affinity chromatography and SDS-PAGE gel electrophoresis,the purified product was stained with Coomassie Brilliant Blue.It was found that the purified product had a single molecular weight of about 17.5 kD.The results of Western Blot confirmed that by ALR antibody.CCK-8 assay showed that rhALR promoted the proliferation of HepG2 cells in a concentration-dependent manner.2.Mouse bone marrow mesenchymal stem cells can be effectively isolated and purified by cell adherence separation assay.Flow cytometry showed that the positive rates of CD105,CD29,CD44 and CD90.2 were more than 95%,while the negative rates of CD34 and CD45 were less than 5%,which accorded with mouse bone marrow mesenchymal cells.Compared with the control group,ALR(1,10,100 ug/mL)at 48 hours promoted the proliferation of bone marrow mesenchymal stem cells(p < 0.01).After 7 days of osteogenesis induction,alkaline phosphatase activity increased;after 14 days of osteogenesis induction,calcium nodules increased and the expression of osteogenesis-related genes(Runx2,OCN,OPN)increased(p < 0.01).Conclusion: ALR might promote the osteogenic differentiation of bone marrow mesenchymal stem cells.Objectives This meta-analysis was designed to investigate the efficacy and safety of PTH in fracture healing.Methods The EMBASE,Pub Med,and Cochrane Library databases were systematically searched from the inception dates to April 26,2018.The primary randomized clinical trials comparing PTH treatment for fracture healing with placebo or no treatment were identified.We did not gain additional information by contacting the authors of the primary studies.Two reviewers independently extracted the data and evaluated study quality.This meta-analysis was executed to determine the odds ratio,mean difference,standardized mean difference,and 95% confidence intervals with randomeffects models.Results In total,8 randomized trials including 524 patients met the inclusion criteria.There were significant differences in fracture healing time,pain relief and function improvement.There were no significant differences in the fracture healing rate or adverse events,including light-headedness,hypercalcemia,nausea,sweating and headache,except for slight bruising at the injection site.Conclusions We determined that the effectiveness and safety of PTH in fracture healing is reasonably well established and credible. |
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