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.

Objective To explore the association of epidermal growth factor receptor(EGFR)mutant non-small cell lung cancer(NSCLC)with tyrosine kinase inhibitor(TKI)-related rash based on network pharmacology and to predict the potential traditional Chinese medicine.Methods Gene chip data of EGFR mutant NSCLC cell lines and normal fibroblasts before and after treatment with erlotinib,a TKI,were collected from Gene Expression Omnibus database.Differentially expressed genes were screened using limma package of R 4.3.2 software.Cross-over target genes were screened using venn package.The differentially expressed genes and cross-over target genes were analyzed using ClusterProfiler package.CoreMine Medical database was used to predict traditional Chinese medicine of the cross-over target genes,and the nature,flavour,and channel tropism were analyzed.The results were verified by molecular docking method.Results A total of 126 cross-over target genes were screened.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the differentially expressed genes and cross-over target genes were enriched in chromosome,spindle and other regions,and were involved in biological processes such as mitosis and DNA replication.These genes were also associated with signaling pathways including DNA replication,lysosomes,and cell cycle.Gene set enrichment analysis results showed that chemokine pathway and nucleotide-binding oligomerization domain-like receptor signaling pathway were activated in the NSCLC cells,and mammalian target of rapamycin signaling pathway and amino acid metabolic pathway were disturbed in the fibroblasts after treatment with erlotinib.CoreMine Medical database predicted that 354 kinds of traditional Chinese medicines were mainly classified as cold,warm and flat,bitter,pungent and sweet,belonging to stomach,lung and liver meridians.Taking Scutellaria baicalensis as an example,molecular docking analysis of experimentally validated target genes and their active components revealed strong binding interactions between the target genes and active components.Conclusion EGFR mutant NSCLC and TKI-related rash have homology in pathogenesis,both involving DNA replication and cell cycle,which provides traditional Chinese medicine medication instruction for patients with EGFR mutant NSCLC and TKI-related rash.

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