A cost-effective method to enhance adenoviral transduction of primary murine osteoblasts and bone marrow stromal cells

We report here a method for the use of poly-L-lysine （PLL） to markedly improve the adenoviral transduction efficiency of primary murine osteoblasts and bone marrow stromal cells （BMSCs） in culture and in situ, which are typically difficult to transduce. We show by fluorescence microscopy and flow cytometry that the addition of PLL to the viral-containing medium significantly increases the number of green fluorescence protein （GFP）-positive osteoblasts and BMSCs transduced with an enhanced GFP-expressing adenovirus. We also demonstrate that PLL can greatly enhance the adenoviral transduction of osteoblasts and osteocytes in situ in ex vivo tibia and calvaria, as well as in long bone fragments. In addition, we validate that PLL can improve routine adenoviral transduction studies by permitting the use of low multiplicities of infection to obtain the desired biologic effect. Ultimately, the use of PLL to facilitate adenoviral gene transfer in osteogenic cells can provide a cost-effective means of performing efficient gene transfer studies in the context of bone research.

Mitogen activated protein kinase signaling pathways participate in the active principle region of Buyang Huanwu decoction-induced differentiation of bone marrow mesenchymal stem cells

Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell differentiation into neurons. Mitogen-activated protein kinase signaling was identified as one of the key pathways underlying this differentiation process. The present study shows phosphorylated extracellular signal-regulated protein kinase and phosphorylated p38 protein expression was increased after differentiation. Cellular signaling pathway blocking agents, PD98059 and SB203580, inhibited extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways respectively, mRNA and protein expression of the neuronal marker, neuron specific enolase, and neural stem cell marker, nestin, were decreased in bone marrow mesenchymal stem cells after treatment with the active principle region of Buyang Huanwu decoction. Experimental findings indicate that, extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways participate in bone marrow mesenchymal stem cell differentiation into neuron-like cells, induced by the active principle region of Buyang Huanwu decoction.

Effects of CpG-ODNs on phenotype and function of monocyte-derived dendritic cells in chronic hepatitis B

AIM:To study the effects of synthetic nonmethylated CpG-containing oligodeoxynucleotides(CpG-ODNs) ,either alone or combined with recombinant Hepatitis B surface antigen(HBsAg) polypeptide,on the phenotype,function,and intracellular signaling pathways of monocyte-derived dendritic cells(DCs) in patients with chronic hepatitis B(CHB) .METHODS:Peripheral blood monocytes isolated from CHB patients and healthy volunteers were induced to be dendritic cells by recombinant human granulocyte-monocyte colony stimulating factor and interleukin-4.The DCs were then treated with CpG-ODNs,CpGODNs/HBsAg,or tumor necrosis factor(TNF)-αfor 18 h.The expression of surface molecules including HLA-DR,CD86,and CD1a in DCs were detected by flow cytometry,and the expression of signal transducers and activators of transcription(STAT1,3,4,5,6) and suppressors of cell signaling(SOCS1,3) were determined by Western blotting assay.In addition,the capacity of DCs to stimulate allogeneic T lymphocytes and the amount of IL-12p70 released from DCs were measured.RESULTS:In the DCs derived from patients with CHB,treatment with TNF-α,CpG-ODNs,or CpG-ODNs/HBsAg,as compared to the vector control,significantly increased the expression of HLA-DR,stimulated the release of IL-12p70,and enhanced the capacity of DCs to stimulate allogenic T lymphocytes.The expressions of STAT1/4/6 and SOCS1/3,but not STAT3/5,were upregulated by TNF-α,CpG-ODNs,and CpG-ODNs/HBsAg.In addition,the expression of CD1a was upregulated only in the presence of both CpG-ODNs and HBsAg.CONCLUSION:The treatment with CpG-ODNs,either alone or combined with HBsAg,has a remarkable stimulatory effect on the impaired phenotype and function of DCs in CHB,possibly by regulating the expression of STAT1,4,6 and SOCS1,3.