| Objectives:Osteoarthritis (OA) is a common disease affecting the living quality of the aged people. The incidence of men is5.6%, while the incidence of women is15%among the people over60years. It brings heavy burden to individuals and society. The release of inflammatory factors such as IL-1and TNF plays an important role in the pathogenic process. The biological function study of these inflammatory factors brought a series of related genes therapy recently and the role of synovial cells had been paid more attention. Inflammatory mediators released from synovial cells may cause cartilage matrix degradation, and aggravate inflammatory changes. IL-1Ra is the most important type of inflammatory mediators. In this experiment, the lentivirus mediating interleukin1receptor antagonist (IL-1Ra) gene was transfected into human synovial cells and the transfection efficiency was tested. This experiment tried to use the efficient and stable expression and secretion of IL-1Ra gene in the synovial cells to antagonize the inflammatory factor IL-1and to inhibit the degradation of cartilage matrix.Methods:Synovial cells of human knee joint were cultured in vitro. The recombinant plasmids expressed by lentivirus mediating IL-1Ra were transfected into human synovial cells in logarithmic growth phase, and the transfection efficiency was observed under the fluorescence microscope. The expression of transfection gene was determined by ELISA test and immunohistochemistry assay, and the effects of gene transfection on the rate of cell proliferation were studied by detecting the cell cycle. Synovium tissues of human knee joint were taken from knee replacement surgery and transported to the laboratory under aseptic conditions. Then synovial cells were primarily cultured and subcultured to the secondary generation (P2generation). The transfection experiments were carried after cells purification. The P2generation cells were divided into three groups:lentivirus mediating IL-1Ra transfection group (group A), lentivirus vector transfection group (group B-negative control group), non-transfection group (group C-blank control group). Using green light as the exciting light, fluorescence states of each group were detected under the fluorescence microscope72hours after transfection, and the transfection efficiency was calculated. The culture supernatants of each group were collected respectively to determine protein expressions of IL-1Ra by ELISA test, and to investigate if the synovial cells transfected by lentivirus mediating IL-1Ra gene can secrete IL-1Ra proteins efficiently by comparing and analyzing the three groups. The cells transfected after72hours were implanted on the glass slides, and placed in6pore plates. Stained with DAB after24hours and counterstained with hematoxylin, the results were observed under the microscope, and the expression rates of IL-1Ra in each group were calculated.Results:Lentivirus mediating IL-1Ra genes can be transfected into synovial cells of human knee joint efficiently. Under the fluorescence microscope, green fluorescence expression was observed strong in the synovial cells transfected after72hours and the transfection efficiency was about90%. The data from ELISA test in the supernatant of synovial cells transfected after72hours showed that the concentrations of IL-1Ra protein in group A, group B, group C were (58.723±3.156) ng/ml,(10.598±3.821) ng/ml, and (11.263±6.833) ng/ml respectively. The immunohistochemistry assay showed that staining of IL-1Ra protein particles in the cytoplasm of about95%cells in group A could be seen clearly, while fluorescent IL-1Ra in supernatants of group B and group C were below the level of detection.Conclusions:By transfecting the lentivirus mediating IL-IRa gene into human synovial cells, we found that the fluorescent expression rate of cells transfected by target gene was high. IL-1Ra protein can be effectively expressed in transfectants and secreted into extracellular to antagonize IL-1and protect articular cartilage, which will provide a theoretical basis for IL-1Ra therapy for osteoarthritis.
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