BACKGROUND:Chitosan is wel known as good biocompatibility and biodegradability;however, its extensive use in biomedical applications is restricted due to its poor transfection efficiency. OBJECTIVE:To prepare the polyethyleneimine-chitosan/DNA nanoparticles loading enhanced green fluorescent protein gene, and to detect their physicochemical properties and gene transfection efficiency towards chondrocytes in vitro.
METHODS:Low molecular weight polyethyleneimine was covalently linked to chitosan backbone to construct chitosan-graft-polyethyleneimine;then the chitosan-graft-polyethyleneimine was mixed with DNA nanoparticles, which loaded enhanced green fluorescent protein gene, by a complex coacervation method. The nanoparticle morphology was observed under a scanning electron microscopy. The sizes and zeta-potentials of the
nanoparticles were measured by a Marven-nano laser diffractometer. The binding capacity of plasmid DNA was evaluated by agarose gel electrophoresis analysis. The gene transfection experiments in vitro were performed towards rabbit’s chondrocytes. The gene transfection efficiency was measured with flow cytometry and under fluorescence microscope. How marked DNA entered into the nucleus of chondrocytes mediated by the nanoparticles was detected by laser scanning confocal microscopy.
RESULTS AND CONCLUSION:The prepared nanoparticles were mainly spherical, with an average size of (154.6±18.6) nm, and zeta-potential of (24.68±6.82) mV. The agarose gel electrophoresis analysis confirmed that the nanoparticles could effectively protect plasmid DNA from DNase Ⅰ-induced degradation. Gene transfection in vitro proved that the nanoparticles were efficient in transfecting rabbit’s chondrocytes and the expression of green fluorescent proteins was observed under fluorescent microscope, with a transfection efficiency of (23.80±1.74)%that was significantly higher than that of the naked plasmid DNA and chitosan/DNA nanoparticles (P<0.05). But no significant differences were observed between polyethyleneimine-chitosan/DNA nanoparticles and LipofectamineTM 2000. These findings indicate that the polyethyleneimine-chitosan/DNA nanoparticles can effectively protect plasmid DNA from nuclease degradation, and exhibit the favorable transfection ability towards articular chondrocytes.

Objective To investigate the cytokine spectrum and henlatopoiesis-supportive function of umbilical cord derived mesdnchymal stem cells(UC-MSC),and compare with those of normal adult bone marrow derived mesenchymal stem cells(BM-MSC).Methods The mRNA of cytokine production of UC-MSC and BM-MSC were determined by reverse transcriptasc polymerase chain reaction(RT-PCR)analysis.To evaluate hematopoiesis supporting activity,cord blood(CB)CD+34 cells were co-cultured with UC-MSC or BM-MSC.Colony-forming cells(CFC)were determined after 5 weeks of culture.Results RT-PCR assay showed that UC-MSC had a cytokine spectrum very similar to that of BM-MSC.including expression of the mRNA ofstem cell factor,leukemia inhibitor factor,macrophage colony stimulating factor,Flt3-ligand,interleukin-6,vascular endothelial growth factor and stromal derived factor-1.but UC-MCS additionally expressed mRNA of granulocyte macrophage and granulocyte colony-stimulating factors.After co-culture with CD+34 cord blood cells for 5 weeks,no significant difference in CFC was observed between the CD+34 cells/UC-MSC and CD+34 cells/BM-MSC co-cultures (P＞0.05). Conclusion The cytokine spectrum and hematopoiesis-supponive function of UC-MSC ale similar with that of BM-MSC.

Essential fatty acids are those that could not be synthesized by the body itself but crucial for health and life. Studies have shown that ω-3 fatty acids may facilitate human physiological functions. Mammals lack ω-3 desaturase gene, and the Δ15 fatty acid desaturase (Δ15 Des) from Caenorhabditis elegans can transform the ω-6 polyunsaturated fatty acids (PUFAs) into ω-3 PUFAs. Transgenic mice expressing Δ15 Des enzyme activity was constructed by using a PiggyBac transposon (PB). Homozygous transgenic mice with stable inheritance was bred in a short time, with a positive rate of 35.1% achieved. The mice were fed with 6% ω-6 PUFAs and the changes of fatty acids in mice were detected by gas chromatography (GC). The expression level of Δ15 Des in mice was detected by quantitative PCR (qPCR) and Western blotting (WB). qPCR and GC analysis revealed that the percentage of positive mice harboring the active gene was 61.53%. Compared with traditional methods, the transformation efficiency and activity of Δ15 Des were significantly improved, and homozygotes showed higher activity than that of heterozygotes. This further verified the efficient transduction efficiency of the PiggyBac transposon system.
Animals ; Caenorhabditis elegans/genetics* ; Fatty Acid Desaturases/genetics* ; Fatty Acids ; Fatty Acids, Omega-3 ; Mice ; Mice, Transgenic