Transfect HAS2 Gene Into Human Temporomandibular Joint Synovial Cells

Temporomandibular joint disorder,TMD is a common disease,which can cause pain and trismus.The inflammation of the synovial membrane is commonly observed.As a result,the synovial fluid changes as well,especially the hyaluronan(HA),a major constituent of synovial fluid at a concentration of 1-4 mg/ml in healthy individuals,which plays important roles in joint lubrication and wear protection.In TMD patients,HA in synovial fluid has lower molecular weight and concentration.Intra-articular injecting HA has been considered an efficient therapy of arthritis in clinical for 30 years,but repeatly intra-articular injecting is indispensable,meanwhile,painful and costly.Particularly it was reported that HAS2 shows much higher enzyme activity and produces the highest molecular weight of HA among common HASs.It is indicate that HAS2-gene-based treatments are capable of efficient in means of providing long-term producing highest molecular weight of HA intra articular.Lentiviral expression of HAS2 and EGFP mediated by FMDV 2A will give a chance to increase the therapeutic efficiency.This study includes 4 parts:Part one:Clone human HAS2 genePurpose:To obtain the complete fragment of human HAS2 gene.Materials and methods:human TMJ synovial fibroblasts were cultured by tissue block method,after extracting the whole genome,PCR complete fragment of HAS2 gene by Suitable primers.After the product was purified by agarose gel electrophoresis,enzyme digestion and gene sequencing were detected.Results:Electrophoresis showed that the size of the fragment was about 1.6kb,consistent with the target fragment size.Enzyme digestion display properly connected with T vector and sequencing showed no error base,matching gene bank correctly.Conclusion:Obtain the complete fragment of human HAS2 gene.Part two:Construction of prokaryotic expression plasmid of EGFP-HAS2 fusion protein,Detection of prokaryotic expression of the gene.Purpose:Added EGFP to the HAS2,for the later experiments can be observed easily.Construct prokaryotic expression plasmid of EGFP-HAS2 fusion protein,Detect prokaryotic expression of the HAS2 gene.Materials and methods:EGFP gene was amplified by PCR from plasmid PUGW.The EGFP gene is connected to the upstream of the HAS2 gene,which is added with the connecting peptide GGGGS.Insert the EGFP-GGGGS-HAS2 gene fragment into the prokaryotic expression plasmid PET-28a,then transformed to E.coli BL21 and induced to express,the total protein was extracted.SDS-PAGE gel electrophoresis,Coomassie brilliant blue staining and Western blot were performed to detect the expression of EGFP-HAS2 fusion protein.Results:SDS-PAGE gel electrophoresis,Coomassie brilliant blue staining and Western blot were not detected in the target band.Conclusion:The expression of EGFP-HAS2 fusion protein in Escherichia coli is difficult.Part three:Construction of eukaryotic expression vector of human HAS2 gene and EGFP mediated by 2A sequencePurpose:Construction of eukaryotic expression vector of human HAS2 gene and EGFP mediated by 2A sequence.Materials and methods:Modify the EGFP-HAS2 connection peptide,changed to T2A sequence,the sequence was changed to HAS2-2A-EGFP by PCR.Insert the target fragment into the eukaryotic expression vector FUGW.The recombinant plasmid and packaging plasmid were cotransfected into 293T cells,and then packaged into lentivirus.Infect human TMJ synovial cells,to observe whether there is fluorescence.Results:Restriction enzyme digestion and electrophoresis showed a successful connection of recombinant plasmid.Fluorescence microscopy showed fluorescence from human TMJ synovial cells infected by lentivirus,the fluorescence intensity increased with the concentration of virus Conclusion:Construct of eukaryotic expression vector of human HAS2 gene and EGFP mediated by 2A sequence Successful.Part four:Detection of HAS2 protein expressionPurpose:Further validation of HAS2 gene through the slow virus mediated,whether it can be expressed in human TMJ synovial cells.Materials and methods:Human TMJ synovial cells were infected with HAS2 and then cultured for three days.By WB,cell immunofluorescence,to detect HAS2 protein expression.Results:WB results showed that both HAS2 and EGFP were expressed respectively,and the intensity of expression increased with the increase of virus titer,Immunofluorescence photography shows the lentivirus infected cells not only green fluorescence and red fluorescence,but the cell climbing piece is difficult to distinguish between the protein is localized in the cytoplasm or cell membrane.Conclusion:Exogenous HAS2 can be expressed in human TMJ synovial cells by lentivirus,fluorescent labels can also express.WB results also demonstrated that 2A's self shearing action in cells.