ABSTRACT
The aim of the present study was to investigate the effects of phosphorylatable nucleus localization signal linked nucleic kinase substrate short peptide (pNNS)-conjugated chitosan (pNNS-CS) mediated miR-140 and IGF-1 in both rabbit chondrocytes and cartilage defects model. pNNS-CS was combined with pBudCE4.1-IGF-1, pBudCE4.1-miR-140, and negative control pBudCE4.1 to form pDNA/pNNS-CS complexes. Then these complexes were transfected into chondrocytes or injected intra-articularly into the knee joints. High levels of IGF-1 and miR-140 expression were detected both in vitro and in vivo. Compared with pBudCE4.1 group, in vitro, the transgenic groups significantly promoted chondrocyte proliferation, increased glycosaminoglycan (GAG) synthesis, and ACAN, COL2A1, and TIMP-1 levels, and reduced the levels of nitric oxide (NO), MMP-13, and ADAMTS-5. In vivo, the exogenous genes enhanced COL2A1, ACAN, and TIMP-1 expression in cartilage and reduced cartilage Mankin score and the contents of NO, IL-1ß, TNF-α, and GAG contents in synovial fluid of rabbits, MMP-13, ADAMTS-5, COL1A2, and COL10A1 levels in cartilage. Double gene combination showed better results than single gene. This study indicate that pNNS-CS is a better gene delivery vehicle in gene therapy for cartilage defects and that miR-140 combination IGF-1 transfection has better biologic effects on cartilage defects.
Subject(s)
Cartilage Diseases/drug therapy , Cartilage, Articular/drug effects , Chitosan/pharmacology , Chondrocytes/drug effects , Insulin-Like Growth Factor I/metabolism , MicroRNAs/metabolism , Peptides/pharmacology , Animals , Cartilage Diseases/metabolism , Cartilage, Articular/metabolism , Chondrocytes/metabolism , Gene Transfer Techniques , Humans , Knee Joint/metabolism , Matrix Metalloproteinase 13/metabolism , Nitric Oxide/metabolism , Rabbits , Synovial Fluid/drug effects , Synovial Fluid/metabolism , Tissue Inhibitor of Metalloproteinase-1/metabolism , Transfection/methodsABSTRACT
We have previously identified a novel Trß isoform (TrßΔ) in the rat, in which a novel exon N (108 bps) was found between exon 3 and exon 4 of TrßΔ, which represents the only difference between TrßΔ and Trß1. In this study, we searched for an elongated Trß2-like subtype with one additional exon N. We successfully isolated the entire mRNA/cDNA of a novel elongated Trß2 isoform via PCR in the rat pituitary gland. The mRNA/cDNA was only 108 bps (exon N) longer than that Trß2, and the extension of the sequence was between exon 3 and 4 of Trß. The whole sequence of this novel Trß isoform has been published in NCBI GenBank (HM043807.1); it is named TRbeta2Delta (Trß2Δ). In adult rat pituitary tissue, quantitative real-time RT-PCR analysis showed that the mRNA levels of Trß2Δ and Trß2 were roughly equal (P > 0.05). We cloned, expressed, and purified the His-Trß2Δ protein [recombinant TRß2Δ (rTRß2Δ)]. SDS-PAGE and western blotting revealed that the molecular weight of rTRß2Δ was 58.2 kDa. Using a radioligand binding assay and an electrophoretic mobility shift assay, rTRß2Δ-bound T3 with high affinity and recognized thyroid hormone response element (TRE) binding sites. Finally, in vitro transfection experiments further confirmed that rTRß2Δ binding T3 significantly promotes the transcription of target genes via the TRE. Here, we have provided evidence suggesting that rTRß2Δ is a novel functional TR isoform.
Subject(s)
Pituitary Gland/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Thyroid Hormone Receptors beta/genetics , Thyroid Hormone Receptors beta/metabolism , Animals , Binding Sites/genetics , COS Cells , Chlorocebus aethiops , Cloning, Molecular , DNA, Complementary/genetics , Ligands , RNA, Messenger/genetics , Rats , Transcription, Genetic/genetics , Transfection/methodsABSTRACT
When studying the altered expression of genes associated with cartilage regeneration by quantitative real-time RT-PCR (RT-qPCR), reference genes with highly stable expression during different stages of chondrocyte developmental are necessary to normalize gene expression accurately. Until now, no reports evaluating expression changes of commonly used reference genes in rabbit articular cartilage have been published. In this study, defects were made in rabbit articular cartilage, with or without insulin-like growth factor 1 (IGF-1) treatment, to create different chondrocyte living environments. The stability and intensity of the expressions of the candidate reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S Ribosomal RNA (18S rRNA), cyclophilin (CYP), hypoxanthine phosphoribosyl transferase (HPRT1), and beta-2-microglobulin (B2M) were evaluated. The data were analyzed by geNorm and NormFinder. B2M and 18S rRNA were identified to be suitable reference genes for rabbit cartilage tissues.
