Effects of chitinase-3-like protein 1 on the expression of inflammatory damage-related molecules in mouse skeletal muscle satellite cells induced by lipopolysaccharide / 中华危重病急救医学

Objective:To explore the potential mechanism of chitinase-3-like protein 1 (CHI3L1) involved in skeletal muscle stem cell injury induced by sepsis.Methods:Six different concentrations of lipopolysaccharide (LPS) were used to stimulate mouse skeletal muscle satellite cells cultured in vitro. Enzyme linked immunosorbent assay (ELISA) and cell counting kit-8 (CCK-8) were used to determine the optimal concentration. The overexpression and interference vectors of CHI3L1 were constructed to transfect skeletal muscle satellite cells, and the transfection efficiency was verified by polymerase chain reaction (PCR) and Western blotting. The cells were randomly divided into blank control group (cells without any intervention), model group (LPS-stimulated untransfected cells), overexpressing CHI3L1 group (LPS-stimulated cells transfected with CHI3L1 plasmid), overexpressing CHI3L1 control group [LPS-stimulated cells transfected with negative control (NC) plasmid], CHI3L1 interference group [LPS-stimulated cells transfected with CHI3L1 small interfering RNA (siRNA)], CHI3L1 interference control group (LPS-stimulated cells transfected with CHI3L1-siRNA NC). The levels of extracellular caspase-1 and interleukin-1β (IL-1β) were detected by ELISA. The protein expressions of intracellular IL-1β, signal transducer and activator of transcription 3 (STAT3), protein kinase B (Akt) and phosphorylated Akt (p-Akt) were detected by Western blotting. Results:According to the results of CCK-8 and ELISA, the best concentration of 5 mg/L LPS was selected for the subsequent experiment. The transfection was validated by PCR and Western blotting. Compared with the blank control group, the levels of extracellular IL-1β, caspase-1 and the protein expressions of intracellular Akt, p-Akt, and IL-1β were significantly increased in the model group [IL-1β (ng/L): 11.22±0.55 vs. 8.63±0.63, caspase-1 (pmol/L): 9.47±0.22 vs. 8.65±0.15, Akt/GAPDH: 1.36±0.12 vs. 1.06±0.15, p-Akt/GAPDH: 0.78±0.07 vs. 0.09±0.01, IL-1β/GAPDH: 1.38±0.12 vs. 0.18±0.03, all P < 0.05]. Compared with the model group and the overexpressing CHI3L1 control group, the levels of extracellular IL-1β, caspase-1 and the protein expressions of intracellular p-Akt and IL-1β were significantly increased in the overexpressing CHI3L1 group [IL-1β(ng/L): 14.93±0.97 vs. 11.22±0.55, 9.38±0.40, caspase-1 (pmol/L): 10.35±0.03 vs. 9.47±0.22, 8.46±0.24, p-Akt/GAPDH: 1.21±0.04 vs. 0.78±0.07, 0.63±0.04, IL-1β/GAPDH: 1.87±0.08 vs. 1.38±0.12, 1.51±0.17, all P < 0.05]. Compared with the model group and the CHI3L1 interference control group, the levels of extracellular IL-1β, caspase-1 and the protein expressions of intracellular p-Akt and IL-1β were significantly decreased in the CHI3L1 interference group [IL-1β(ng/L): 8.98±0.73 vs. 11.22±0.55, 10.44±0.65, caspase-1 (pmol/L): 7.61±0.63 vs. 9.47±0.22, 8.37±0.38, p-Akt/GAPDH: 0.50±0.04 vs. 0.78±0.07, 0.94±0.06, IL-1β/GAPDH: 0.77±0.02 vs. 1.38±0.12, 1.13±0.07, all P < 0.05]. Conclusions:CHI3L1 may mediate the damage of skeletal muscle stem cells in sepsis by increasing the expression of caspase-1 and IL-1β. CHI3L1 may be involved in the regulation of Akt signaling pathway in skeletal muscle stem cells, but has no significant effect on STAT3 signaling pathway.

In Vitro and In Vivo Osteogenesis of Human Orbicularis Oculi Muscle-Derived Stem Cells

BACKGROUND: Cell-based therapies for treating bone defects require a source of stem cells with osteogenic potential. There is evidence from pathologic ossification within muscles that human skeletal muscles contain osteogenic progenitor cells. However, muscle samples are usually acquired through a traumatic biopsy procedure which causes pain and morbidity to the donor. Herein, we identified a new alternative source of skeletal muscle stem cells (SMSCs) without conferring morbidity to donors. METHODS: Adherent cells isolated from human orbicularis oculi muscle (OOM) fragments, which are currently discarded during ophthalmic cosmetic surgeries, were obtained using a two-step plating method. The cell growth kinetics, immunophenotype and capabilities of in vitro multilineage differentiation were evaluated respectively. Moreover, the osteogenically-induced cells were transduced with GFP gene, loaded onto the porous β-tricalcium phosphate (β-TCP) bioceramics, and transplanted into the subcutaneous site of athymic mice. Ectopic bone formation was assessed and the cell fate in vivo was detected. RESULTS: OOM-derived cells were fibroblastic in shape, clonogenic in growth, and displayed phenotypic and behavioral characteristics similar to SMSCs. In particular, these cells could be induced into osteoblasts in vitro evidenced by the extracellular matrix calcification and enhanced alkaline phosphatase (ALP) activity and osteocalcin (OCN) production. New bone formation was found in the cell-loaded bioceramics 6 weeks after implantation. By using the GFP-labeling technique, these muscle cells were detected to participate in the process of ectopic osteogenesis in vivo. CONCLUSION: Our data suggest that human OOM tissue is a valuable and noninvasive resource for osteoprogenitor cells to be used in bone repair and regeneration.

Study of repair of bone defect using rabbit skeletal muscle stem cells transduced by AdrhBMP-2 gene / 中国康复理论与实践

@#ObjectiveTo observe the effect of rabbit skeletal muscle stem cells (RSMSCs) modified by adenovirus mediated bone morphogenetic protein-2 (BMP-2) gene ex vivo in combination with demineralized bone matrix (DBM) on repair of longer bone defect in rabbit.MethodsModel of radial bone defects (20 mm) of rabbits was established. 50 rabbits were divided into 5 groups, group A (AdrhBMP-2 trusduced RSMSCs/DBM group), group B (adGFP trusduced RSMSCs/DBM group), group C (not trusduced RSMSCs/DBM group), group D (DBM group), and group E (untreated group). Roentgenographic, histologic, biomechanical, bone density of all animals were examined at the end of 4th and 6th week after surgery.ResultsAt 4th week, radial bone defects healed in group A. The healing rates from group A to group E were 100%, 50%, 33%, 0%, and 0% respectively at 6th week.ConclusionRSMSCs modified by AdrhBMP-2 ex vivo in combination with DBM can repair radial longer segemental defect in rabbit. It's possible to be used in the clinical treatment of longer segemental bone defect.

Osteoinductivity of recombinent human bone morphogenetic protein-2 gene mediated with cationic liposome transfer in skeletal muscle stem cells of rabbits in vitro / 中国康复理论与实践

@#ObjectiveTo evaluate the osteoinductivity of cationic liposome mediated recombined human bone morphogenetic protein-2 gene (rhBMP-2) transfer in skeletal muscle stem cells (SMSCs) of rabbits. MethodsSMSCs were transfered by pAGFP rhBMP-2 liposome complex. Gene transferred SMSCs were evaluated with immunohistochemistry.ResultsThe transfection rate of SMSCs transferred by cationic liposome mediated pAGFP-rhBMP-2 was 14.18%. Positive stain of rhBMP-2, alkaline phosphatase(ALP),collegenⅠhad been found in the transferred SMSCs.ConclusionQuantity of SMSCs is enough to be target cells of gene therapy. Cationic liposome can mediated bone morphogenetic protein-2 gene transfected SMSCs in vitro. Compared with AdrhBMP-2,the transfection rate of liposome mediated bone morphogenetic protein-2 gene transfected SMSCs is lower.