Recently, specific miRNAs were reported to be involved in chondro

Recently, specific miRNAs were reported to be involved in chondrogenesis and inflammatory cartil age diseases. MiR 675 regulates type II collagen in articular chondrocytes, miR 18 regulates chodnrocytic phenotype by targeting Ccn2/Ctgf. Despite consider they able evidences regarding the involvement of miRNAs in cartilage development, identifications and func tions of miRNAs in cartilage development/degeneration Inhibitors,Modulators,Libraries are poorly understood. In the present study, to better understand the molecu lar mechanisms involved in the OA pathology, we iden tify miRNAs from normal and OA chondrocytes and characterize the functional role of miRNA 488, which could have important diagnostic and therapeutic potential.

Methods Primary cultures of human Inhibitors,Modulators,Libraries chondrocytes Human chondrocytes were prepared from macroscopic ally severely damaged zones of osteoarthritic knee joints obtained undergoing total knee replacement or biopsy of normal cartilages. The study was carried out in full ac cordance with Wonkwang University ethics guidelines and cartilage samples were collected Inhibitors,Modulators,Libraries after obtaining writ ten informed consent of the donors. Cartilage small slices were sequentially digested with 0. 06% collagenase then seeded at a density of 1. 5 104 cells/cm2 in culture medium consisting of DMEM supple mented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 ug/ml streptomycin. RNA preparation and miRNA real time PCR Total Inhibitors,Modulators,Libraries RNA was isolated using the mirVana miRNA iso lation kit. miRNA analysis was performed using RT2 miRNA PCR Arrays and individual miRNA expression were independently quantified using the TaqMan MicroRNA, according to the manufacturers protocols.

Production of lentiviral particles The hsa miR 488 and Negative Control lentivirus was transfected with 3rd generation packaging mix from Ap plied Biological Materials Inc. into Inhibitors,Modulators,Libraries human 293FT cells using Lentifectin in Opti MEM I medium and cultured overnight. The super natant was collected and lentiviral particles were con centrated using Lenti X Concentrator. Experimental OA and histology of OA cartilage Experimental OA was induced by destabilization of the medial meniscus surgery 8 week old male mice. Sham operated animals injected with empty lentiviruses were used as controls for DMM. Mice were killed selleckchem 8 weeks after DMM surgery or 2 weeks after intraarticular injection of si ZIP 8 expressing lentiviruses for histo logical and biochemical analyses. Cartilage destruction in mice was examined using Safranin O staining. Briefly, knee joints were fixed in 4% paraformaldehyde, decal cified in 0. 5 M EDTA for 14 days at 4 C, and embedded in paraffin. The paraffin blocks were sec tioned at 6 um thickness. The sections were deparaffinized in xylene, hydrated with graded ethanol, and stained with Safranin O.

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