Killing Action Of Adenovirus Vector Carrying HSV-TK/GCV System On Synoviocytes Of Pigmented Villonodular Synovitis

ObjectivePigmented villonodular synovitis is a kind of proliferative disease of synovial membrane,bursa synovialis and tendinous sheath.Its feature is proliferation of synovial membrane,villonodular formation,haemosiderin deposition. It is a synovial disease between inflammation and benign tumor which is invasive to joint tissue.It is insensitive to traditional therapy.Gene therapy,especially suicide gene,is more and more used in clinical study.HSV-TK/GCV is the most common suicide gene system.The gPVNS1 of our study is to evaluate the effect of HSV-TK/GCV on synoviocytes of PVNS and explore effective gene therapy of PVNS.Material and methods1 Adenovirus with green fluorescent protein were purchased from Vector Gene Technology Company Limited and reserved in central laboratory of our hospital, whose titer was3.2×10¹¹pfu/ml.GCV was purchased from Hubei Keyi Pharmacy Company Limited.2 The synovial membrane was collected from the patient with PVNS when the operation 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 transfected 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 total RNA was extracted from 1×10⁴ transfected cells,then PCR was done according to the HSV1 template,and expression of HSV-TK was detected.4 The synoviocytes of third generation were cultured on the tissue culture plates with 96 holes,and transfected by 120 MOI adenovirus,compared with untransfected cells.GCV of different concentration were added after 24 hours, then after 48 hours,optical density was determined after we added MTT for 4 hours.Survival ratio was determined according to the formula.Curve of survival rate was drawed.5 Cells tranfected with 120 moi adenovirus mixed with untransfected cells for 24 hours on the tissue culture plates according to rate of 10%,25%,50%,75%, compared with uninfected cells,survival ratio of cells was calculated after we added GCV(10μg/ml)to observe the bystander effect.6 synoviocytes were have been infected for 24hours by AD-TK and added GCV(10μg/ml),and then we can observe the changes of configuration of synoviocytes with the phase contrast microscope at 12h,24h,and 48h respectively. After synovial cells were infected for 24 hours by AD-TK and added GCV(10μg/ml)for 48 hours,the dyed and fixuped by Hoechst33342/PI, synoviocytes were dropped on slide,added cover glass,observed and counted with fluorescent microscope.7 Synoviocytes were transfected for 24 hours and added GCV of 10μg/ml for 48 hours.Then we digested,centrifuged and collected all of synoviocytes,added Annexin V FITC and PI,measured with flow cytometry.Results1 After synoviocytes adhered the wall,two kinds of cells can be observed with the microscopy,namely macrophages-like cell(MCs)and fibroblast-like cells(FCs). When these cells were cultivated to the third generation,FCs were the main cells. FCs was fusiforrn shape and proliferate active.Synovium cell expressed GFP after transfected by adenovirus;when the concentration of the adenovirus was above 120 moi,all the synoviocytes were transfected.2 Synovium cell expressed GFP after transfected by adenovirus;when the concentration of the adenovirus was above 120 moi,all the snoviocytes were transfected.3 1.5%agarose gel electrophoresis by the PCR product shows a specific strap, which is about 1.13kb,compared with the untransfected group.That demonstates that HSV-TK gene was integrated into the gene of synoviocytes.4 The synoviocytes tranfected with 120 moi adenovirus was added GCV,which are different concentrations.The concentration of GCV was more high,the survival rate of cells were more slow.10 ug/ml GCV could kill 80%of transfected cells, while 100 ug/ml GCV could only kill few untransfected cells.So Ad-tk/GCV is effective to transfected synoviocytes.5 The transfected cells were mixed together with untransfected cells,then GCV of 10 ug/ml was added.The survival rate of cells are related to the propotion of the tranfected cells.When the transfected cells took up 75%,almost all the cells died. So the bystander effect is obvious.6 After synoviacytes 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 dim;48h later,part of cells were schizolysis to fragments.Dealed with HSV-TK/GCV,synoviocytes were divided into three states after double dyed by Hoechst33342/PI.Normal cells became light blue,apoptosis cells were thick dying or granulo-like,necrotic cells were red respectively.7 Flow cytometry measure:Comparatively a large percent of cells were necrotic or apoptosis after they have been transfected by TKgene and cultivated with GCV for 48 hours,the percentage of necrosis and apoptosis was 58.7%and 23.4% respectively,but there was no evident necrosis or apoptosis in control group.Conclusion1 Adenovirus vector carrying HSV-TK could transfect synoviocytes of PVNS cultured in vitro highly.2 HSV-TK/GCV system could have a depressive effect on synoviocytes of PVNS cultured in vitro.GCV had concentration dependence within a definite range. HSV-TK/GCV system had a bystander effect on synoviocytes.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 have a depressive effect on synoviocytes cultured in vitro with flow cytometry,and the rate of necrosis was higher that of apoptosis.