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Objective To develop a suitable medium and optimize culture time for the primary osteoblast culture from suckling mouse,so to provide an improved experimental protocol for primary osteoblast culture in vitro.Meth-ods Primary osteoblasts were collected from skull of CD1 suckling mouse by interrupted enzyme digestion.The pu-rified osteoblasts were harvested by differential centrifugation.The incubation time,concentration of fetal bovine se-rum(FBS),β-glycerophosphate sodium and dexamethasone were tested and optimized.The change of osteoblast maturation marker was examined by Western blot(WB)and immunofluorescence staining(IF).The osteogenic ac-tivity was determined by alkaline phosphatase staining,alizarin red staining and ultrastructure.Results Primary osteoblast were obtained from sucleling mouse skull bone by interrupted enzyme digestion for proliferation and trans-generational expansion.The expression of osteoblast maturation markers was parallel to the time of induction culture and the concentration of FBS.Mature osteoblasts were obtained by culturing the cells with 10％ FBS for 14 days.The differentiation of primary osteoblasts was induced by different concentrations of β-glycerophosphate and dexam-ethasone.The results showed that the expression of osteoblast maturation markers was higher under the culture con-ditions of 10 mmol/L β-glycerophosphate and 5 nmol/L dexamethasone(P<0.01),and the staining of alkaline phosphatase and alizarin red was obvious,and the osteogenic activity was better too.Conclusions Primary osteo-blasts isolated from the skull of suckling CD1 mice cultured in induction medium containing 10％fetal bovine ser-um,10 mmol/L β-glycerophosphate sodium and 5 nmol/L dexamethasone for 14 days show good osteogenic activity and are suitable for in vitro experimental studies.
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[ ABSTRACT] AIM: To investigate the expression and significance of Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) in chronic superficial gastritis.METHODS:The Wistar rats (n=30) were randomly divided into blank group, model group and control group.The Wistar rat model of chronic superficial gastritis was established by in-tragastric administration of 0.02%ammonia and long-term irregular diet.All rats were sacrificed, and gastric tissues were harvested and stained with hematoxylin and eosin.The expression of Lgr5 at mRNA and protein levels was analyzed by re-verse transcriptional polymerase chain reaction, Western blotting and immunohistochemistry.RESULTS:Lgr5 was mainly expressed in the cell membrane and cytoplasm.Lgr5 showed high expression in model group compared with blank group and control group.No obvious difference between blank group and control group was observed.CONCLUSION:Persistent in-flammation leads to increased expression of Lgr5.Lgr5 may be a proinflammatory tumor promoting factor.
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This study sought to probe into the mechanism of spontaneous contraction of portal vein. The morphological and electrophysiological characteristics of the freshly isolated interstitial cells (ICs) of rabbit portal vein were investigated by using immunohistochemical and conventional whole-cell patch clamp techniques. The isolated interstitial cells exhibited stellate-shaped or spindle-shaped bodies with a variable number of thin processes projecting from cell bodies, and these cells were noted to be c-Kit immunopositive. Under conventional whole-cell patch clamp configuration, the membrane potential was held at -60 mV, the spontaneous rhythmic inward currents were recorded in ICs, and the frequencies of which were similar to those of spontaneous contraction of portal vein. The inward currents were insensitive to nicardipine (an L-type calcium channel blocker) but could be abolished by gadolinium (a non-selective cation channel blocker). The results suggested that the spontaneous rhythmic inward currents recorded in freshly isolated ICs may be pacemaker currents which elicit the spontaneous contraction of portal vein.