| ObjectiveOsteoarthritis(OA) is a common disease in the field of orthopedics,and 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.The synovium's inflammatory hyperplastic reaction is an important participant which destroyed the articulation and accelerated the course of OA.Osteoarthritic synoviocytes hyperplasy and become active under ultrastructural observation.In addition,IL-1 is the most important cytokines in the pathological process of OA,which leads to articular cartilage degradation and destruction.Up to now the therapeutic efficacy and convenience of OA are unsatisfactory.The research of gene therapy about OA is in progress and encourages its treatment.For pathological synovial proliferation and the abnormal secretion of the proinflammatory factor IL-1 in articular cavity,we use HSV1-tk/GCV to inhibit the proliferation of synovial cell,use its "bystander effect" to kill the synovial cells which have not been transfected,and use hIL-1Ra to against the role of proinflammatory factor IL-1.The goal of our study is to construct adeno-associated virus(AAV) vector plasmid pSNAV2.0 co-expressing the herpes simplex virus 1 thymidine kinase (HSV1-tk) gene and human interleukin-1 receptor antagonist(hIL-1Ra) gene by utilizing an internal ribosome entry site(IRES) sequence to link the two cDNAs,and to detect its expression in synovial cells,which will provide a sound base for gene therapy of osteoarthritis.Material and methods1 pORF-HSV1-tk was purchased from InvivoGen;pIRES2-EGFP was purchased from Clontech;pCDI-hIL-1Ra is from Professor Ma Dalong favors.2 The primers were designed with the restriction endonuclease positions according to multiclone sites sequence of the pSNAV2.0 plasmid.The HSV1-tk,hIL-1Ra and IRES were amplified by the polymerase chain reaction(PCR).3 The HSV1-tk,hIL-1Ra and IRES,which were amplified by PCR,were inserted into pMD18-T simple vector respectly to construct pMD-HSV1-tk,pMD-IRES and pMD-hIL-1Ra.Measure gene sequences.4 After being verified by sequencing,the hIL-1Ra and HSV1-tk were cloned into pMD-IRES plasmid to construct the pMD-HSV1-tk-IRES-hIL-1Ra.The recombinant plasmid was identified by Kpnâ… .5 The HSV1-tk-IRES-hIL-1Ra fragment was then cloned into the adeno-associated virus(AAV) vector plasmid pSNAV2.0.The recombinant expression plasmid pSNAV2.0-HSV1-tk-IRES-hIL-1Ra was constructed and identified by Kpnâ… .6 The synovial cells was collected from a patient with OA when the operation was done,and centrifuged,cultured primarily,and then generated.7 pSNAV2.0-HSV1-tk-IRES-hIL-1Ra was transfected into synovial cells with Lipofectamine 2000 method.The mRNA level change of HSV1-tk-IRES-hIL-1Ra was identified by the reverse transcription polymerase chain reaction(RT-PCR).Results1 The HSV1-tk,hIL-1Ra and IRES were amplified by PCR.The size of amplified products were 1153bp,622bp and 546bp,which were consistent with the expected.2 pMD-HSV1-tk,pMD-IRES and pMD-hIL-1Ra were constructed.The Sequencing results of HSV1-tk,hIL-1Ra and IRES were Consistent with GenBank.3 pMD-HSV1-tk-IRES-hIL-1Ra was identified by Kpnâ… .The result was consistent with the expected.4 pSNAV2.0-HSV1-tk-IRES-hIL-1Ra was identified by Kpnâ… .The result was consistent with the expected.The recombinant plasmid was successfully constructed.5 After synoviocytes adhered the wall,three kinds of cells can be observed with the microscopy,namely dendritic-like cells(DCs),macrophages-like cells(MCs) and fibroblast-like cells(FCs).When these cells were cultivated to the third generation, FCs were the main cells.FCs were fusiform shape and proliferate active.6 The RT-PCR result indicated that HSV1-tk and hIL-1Ra were effectually expressed in the transfected synovial cells.ConclusionThe recombinant plasmid pSNAV2.0-HSV1-tk-IRES-hIL-1Ra,which would be packed in AAV,was successfully constructed and effectually expressed in the transfected synovial cells,which provided a sound base for gene therapy of osteoarthritis.
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