Effect Of Suicide Gene HSV-TK/GCV System With Adenovirus Mediation On Proliferous Osteoarthritic Synoviocytes

ObjectiveOsteoarthritis is a common disease in the field of orthopedics,and the synovium's inflammatory hyperplastic reaction is an important participant which destroyed the articulation and accelerated the course of osteoarthritis. Osteoarthritic synoviocytes hyperplasy and become active under ultrastructural observation. It does a great harm to the health of the aged and becomes an important factor leading to losing the ability to work in the long term. Up to now the therapeutic efficacy and convenience of osteoarthritis are unsatisfactory.The research of gene therapy about osteoarthritis is in progress and encourages its treatment. The goal of our study is to evaluate the effect of herpes simplex virus-thymidine kinase/ganciclovir system with adenovirus-mediation on osteoarthritic synoviocytes with suicide gene's mechanism of selective effect on proliferative cells. Materials and Methods1. Adenovirus with green fluorescent protein were reserved in central laboratory of our hospital. Amplification, purification and titer determination of Ad CMV HSV-TK were finished and its titer was 1×10¹².2. The synovial membrane was collected when total knee arthroplasty of osteoarthritis was done, and centrifuged by Goto separating medium, cultured primarily, and then generated. The synoviocytes of third generation were cultivated on the tissue culture plates and transduced with the adenovirus containing HSV-TK gene of different titers for 24 hours;The first hole of PBS as blank control group.The expression of GFP taked by cells was showed by fluorescent microscopy.The infection rate was calculated and the curve was drawed.3. The synoviocytes were cultured on the tissue culture plates and infected by 100 moi adenovirus for 24 hours,compared with uninfected cells.GCV of different concentration were added after 48hours. Optical density was determined after we added MTT for 4 hours. Inhibition ratio of cells was calculated according to the formula.Cells infected with 100 moi adenovirus mixed with uninfected cells for 24 hours on the tissue culture plates according to rate of 20%, 40%, 60%, 80% , compared with uninfected cells. Inhibition ratio of cells was calculated after we added GCV(10mg/l) for 48 hours by the MTT.4. Synoviocytes were have been infected for 24 hours by Ad-TK of 100 moi and added GCV of 10 mg/l,and then we can observe the changes of configuration of synoviocytes with the phase contrast microscope at 0 h ,12 h ,24 h ,48 h , respectively; after Synovium cell was infected for 24 hours by Ad-TK of 100 moi and added GCV of 10 mg/l for 48h,then dyed and fixuped by Hoechst 33342/PI, synoviocytes were dropped on slide,added cover glass,observed and counted with fluorescent microscope.5. Synoviocytes were infected 24 hours by Ad-TK of 100 moi and added GCV of 10 mg/l for 48h.Then digested, centrifuged and collected all of synoviocytes, added Annexin V-FITC, PI; measured with flow cytometry.Results1. After synoviocytes adhered the wall, three kinds of cells can be observed with the microscopy, namely dendritic-like cell(DCs), macrophages-like cell(MCs) and fibroblast-like cells(FCs). FCs were the main components. When these cells were cultivated to the third generation, there was only one type, FCs left. FCs was fusiform shape and proliferate active. Synovium cell expressed GFP after transduced by adenovirus;when the concentration of the adenovirus was above 100 moi, all the snoviocytes were transduced. 2. Measure inhibitory activity of snoviocytes by MTT: the growth of cells was not inhibitory if we only add Ad-TK or GCV;but adding GCV to the group of transduced by Ad-TK(100 moi), cell growed restricted were restricted to grow and with the augment of concentration of GCV the inhibitory activity would be bigger. Inhibitory activity would reach 76.0% if we added GCV(10 mg/l).The infected cells were mixed together with OA synoviocytes.Then GCV of 10 mg/l was added for 48h.When the infected cells took up 20%, inhibitory activity was 31.2% of control group. inhibitory activity of mixed was higher than not.3. After snoviocytes were acted by HSV-TK/GCV system, with phase contrast microscope we discovered that cells became roundness from fusiform shape, within endochylema vacuole emerged, and cell nucleus became larger;24h later,the cells continued to be swollen up and became even larger than before, vacuole became numerous , and cell nucleus became dim;48h later part of cells were schizolysis to fragments. Dealed with HSV-TK/GCV, snoviocytes were divided into three states after double dyed by Hoechst 33342/ PI,. Normal cells became light blue,apoptosis cells were thick dying or granulo-like, necrotic cells were red, respectively.4. Flow cytometry measure Comparatively a larger percent of cells were necrotic or apoptosis after they have been transduced by TK gene and cultivated with GCV for 48h, the percentage of necrosis and apoptosis was 59.1% and 13.4% respectively, but there was no evident necrosis or apoptosis in control group.Conclusion1. It was convenient to draw the materials from OA snoviocytes, and also easy to culture in vitro, passage and amplification; the adenovirus carrier of GFP-phore could infect snoviocytes cultured in vitro highly.2. The HSV-TK/GCV system could have a depressant effect on OA snoviocytes cultured in vitro, GCV had concentration dependence within a definite range; HSV-TK/GCV system had a "bystander effect" on OA snoviocytes. 3. Morphology of cells had changed after acted on by the HSV-TK/GCV system: cell metamorphosed,structure changed,and finally died;cell appeared necrosis or apoptosis after double dyed by fluorescent microscope.4. It was confirmed that the HSV-TK/GCV system could make have a depressant effect on snoviocytes cultured in vitro with flow cytometry,and the rate of necrosis was higher than that of apoptosis.