Over-expression Of Synoviolin Facilitates The Formation Of A Functional Synovial Biomembrane

1. Background:The formation of peritendinous adhesions following the repair of an injury to thedigital flexor tendons remains a major problem in hand surgery. Currently, extrasynovialtendon grafts are frequently used in clinical settings to bridge flexor tendon defects.However, the healing process is always accompanied by postoperative adhesion. This ismostly due to the fact that no synovial membrane tissue covers the extrasynovial tendonsurface, in contrast to the intrasynovial tendon. The synovial membrane plays a veryimportant role in tendon healing and adhesion prevention. It suppresses the extrinsicpathway by preventing contact between the tendon and the exogenous tissues, while itenhances the intrinsic pathway by secreting nutrients to the tendon. For this reason, wehave continued to search for a simple and efficient method to create a functional synovialbiomembrane around extrasynovial tendons.Synoviolin, which is also known as Hrd1, is a novel E3ubiquitin ligase that isimplicated in endoplasmic reticulum-associated degradation. It is an ER-resident membraneprotein with an RING-H2motif. It was cloned from rheumatoid synovial cells and is highlyexpressed in synoviocytes of patients with rheumatoid arthritis (RA),which promotes theoverproliferation of synoviocytes through its anti-apoptotic effect. Due to the ability ofsynoviolin to promote the proliferation of synoviocytes, we conjectured that implantation ofsynoviocytes expressing synoviolin around the extrasynovial tendon might form a synovialbiomembrane on the tendon surface and thus improve tendon healing and prevent adhesion.Our object is to identify the proliferative effect of synoviolin on synoviocytes, andexplore if it is feasible and effective to create a functional synovial biomembrane aroundextrasynovial tendons using synoviocytes overexpressing synoviolin.This project was supported by the grants from National Natural Science Foundation of China (NSFC, No.30672122) 2. Materials and methods:2.1Construction of lentivirus vectorThe lentiviral expression vector with Synoviolin and EGFP was constructed withGateway technology. The plasmid, pLenti4/TO/V5-DEST/Synoviolin-egfp, was thenco-transfected into293FT cells with ViraPower Packaging Mix. The virus wereconcentrated at25,000rpm for20min at4°C. The titers of synoviolin-expressing viruswere determined by infection of293T cells using serial dilutions.2.2Overexpression of synoviolin promotes cellular proliferation and HA secretion ofsynoviocytes.Rabbit synoviocytes were harvested by enzyme digestion and identified byImmunohistochemistry. After infected by Lenti-synoviolin,the mRNA and protein levelwere detected by RT-PCR and Western Blot. Cellular proliferation and increased hyaluronicacid secretion were confirmed in the synoviolin over-expressing synoviocytes byMTT-based method, cell cycle assays and ELISA.2.3Overexpression of synoviolin facilitates the formation of a functional synovialbiomembrane.The extrasynovial tendons were co-cultured with synoviocytes infected byLenti-synoviolin or Lenti-lacZ. The formation of synovial biomembrane was observed byHE staining and SEM; Alcian blue staining was used to detect the secretion of hyaluronicacid by the cultured biomembrane.3. Results:3.1Construction of lentivirus vector3.1.1The cDNA of synoviolin with restriction sites SalI and BamHI was generatedby PCR;3.1.2The cDNA of synoviolin was TA-cloned into pMD18-T simple vector;3.1.3The cDNA of synoviolin was subcloned into the multiple sites of pIRES2-EGFP vector；3.1.4The segment containing the synoviolin-egfp gene was cloned into the multiplecloning site (MCS) of the pENTR1A vector to obtain the entry clone pENTR1A-syno-egfp;3.1.5An LR gateway reaction was used to transfer synoviolin-egfp from thepENTR1A-syno-egfp entry plasmid into the pLenti4/TO/V5-DEST destination vector. 3.1.6The lentiviral expression vector was co-transfected into293FT cells withViraPower Packaging Mix, and the virus was collected48h after transfected;3.1.7The virus was concentrated and the titers of virus were determined by infectionof293T cells using serial dilutions; the titer of Lenti-synoviolin was1×108TU/mL.3.2Overexpression of synoviolin promotes cellular proliferation and HA secretion ofsynoviocytes.3.2.1Isolation and characterization of synoviocytesThe primary cultured synoviocytes were were identified based on morphology afterhematoxylin and eosin (H&E) staining and immunohistochemistry. Cells present spindle orcolumn aspect, the nucleus was orbicular-ovate in the cell center. Immunohistochemistryshowed that cells were positive to vimentin and negative to CD14and CD68, confirmingthe cell type of synoviocytes.3.2.2Synoviocytes overexpressed synoviolin after infected by Lenti-synoviolinAfter being infected with lentivirus, the over-expression of synoviolin in synoviocyteswas confirmed by RT-PCR and western blotting.3.2.3Overexpression of synoviolin promotes cellular proliferation and alters cellcycle in synoviocytes.Cellular proliferation was confirmed in the synoviolin over-expressing synoviocytesby MTT-based method. The cell cycle of was synoviolin over-expressing synoviocytesdetected by flow cytometry, the percentage of cells in G1phase is less thancontrol(P<0.05), and the percentage of cells in S phase is significantly higher thancontrol(P<0.01), indicating that overexpression of synoviolin promotes cellularproliferation in synoviocytes.3.2.4Overexpression of synoviolin promotes HA secretion in synoviocytesTo determine the HA secretion of synoviocytes overexpressing synoviolin,synoviocytes were first infected with lenti-synoviolin and then analyzed using a Rabbit HAELISA Kit to measure the HA concentration in cell supernatants. The results showed that,the HA level was significantly increased at24,48(P <0.05) and72hours (P <0.01) fterbeing infected with lenti-synoviolin. It is indicated that overexpression of synoviolinpromotes HA secretion in synoviocytes.3.3Overexpression of synoviolin facilitates the formation of a functional synovial biomembrane3.3.1Overexpression of synoviolin facilitates the formation of a synovialbiomembraneThe extrasynovial tendons were co-cultured with synoviocytes infected byLenti-synoviolin or Lenti-lacZ. After being co-cultured in vitro for3and7days, theformation of synovial biomembrane was observed by HE staining and SEM; Statisticalevaluation of cell numbers showed that synoviocytes infected by lenti-synoviolin reachedhigher numbers than those infected by lenti-lacZ. These resules indicates thatoverexpression of synoviolin facilitates the formation of a synovial biomembrane.3.3.2The tendon co-cultured with synoviocytes produces HA.Alcian blue staining with Scott’s method was positive at the0.05M MgCl2concentration but negative at the1,2.61, and3.56M MgCl2concentrations for bothlenti-synoviolin-and lenti-lacZ-infected synoviocytes cultured with extrasynovial tendonafter three days. This indicates the presence of HA being synthesized by cells on the tendonsurface.4. Conclusion:4.1Overexpression of synoviolin promotes cellular proliferation of synoviocytes;4.2Overexpression of synoviolin promotes HA secretion of synoviocytes;4.3Overexpression of synoviolin facilitates the formation of a functional synovialbiomembrane.