Objective To investigate the possibility of gene therapy of osteolysis around artificial joint prosthesis by constructing the recombinant adenovirus which can silence tumor necrosis factor α (TNF-α). Methods The primer of small interfering RNA (siRNA) coding sequence of silent TNF-α was designed and amplified, and then RAPAD adenovirus packaging system was used to load the sequence to adenovirus, and the recombinant adenovirus Ad5-TNF-α-siRNA-CMVeGFP which lacked both E1 and E3 regions was constructed. Then 64 female BABL/C mice (weighing, 20-25 g) were randomly divided into 4 groups (n=16): blank control (group A), positive control (group B), simple adenovirus (group C), and treatment group (group D). The prosthetic-model was established in group A, and the prosthetic-loosening-model in groups B, C, and D. At 2 weeks after modeling, PBS solution was injected first, and then the same solution was injected 24 hours later in group A; titanium particle solution was injected, and then PBS solution, Ad5 E1-CMVeGFP (1 × 109 PFU/mL), and Ad5-TNF-α-siRNA-CMVeGFP (1 × 109 PFU/mL) were injected, respectively in groups B, C, and D 24 hours later, every 2 weeks over a 10-week period. The general condition of mice was observed after operation. The tissues were harvested for histological observation, and the expression of TNF-α was detected by Western blot at 12 weeks after operation. Results The positive clones were achieved by enzyme digestion and confirmed by DNA sequencing after loading the target genes into adenovirus vector, and then HEK293 cells were successfully transfected by recombinant adenovirus Ad5-TNF-α-siRNA-CMVeGFP. All mice survived to the completion of the experiment. Histological observation showed that there were few inflammatory cells and osteoclasts in group A, with a good bone formation; there were a large number of inflammatory cells and osteoclasts in groups B and C, with obvious bone destruction; inflammatory cells and osteoclasts in group D was less than those in groups B and C, with no obvious bone destruction. Significant difference was found in the limiting membrane thickness and the number of osteoclasts (group A lt; group D lt; group B lt; group C, P lt; 0.05). Western blot showed that the TNF-α expression levels were 0.235 ± 0.022, 0.561 ± 0.031, 0.731 ± 0.037, and 0.329 ± 0.025 in groups A, B, C, and D respectively, showing significant difference among 4 groups (P lt; 0.05). Conclusion The recombinant adenovirus for silencing TNF-α is successfully constructed, which can effectively inhibit osteolysis by silencing TNF-α expression in the tissues around prosthesis in mice.
Objective To investigate the role of β-catenin in pathogenesis and progression of knee primaryosteoarthritis (OA) by detecting the expression of β-catenin. Methods Between October 2010 and May 2011, 40 cartilagespecimens were collected from adult knee primary OA patients undergoing total knee arthroplasty and 10 cartilage specimensfrom adult patients suffering from amputation and femoral condylar fracture. All cartilage samples were taken out from femoralcondylar. The decalcified paraffin-embedded sections were prepared and stained with fast green-safranin O to observe thedegeneration of cartilage, then the modified Mankin scale was used to classify the degeneration. The expression of β-cateninwas detected by the immunohistochemistry staining and Western blot. Results According to the Mankin scale, 10 caseshad normal cartilage, 12 had mild degenerative cartilage, and 28 had moderate to severe degenerative cartilage. The histologicalobservation showed the mild degenerative cartilage characterized by fissures in the superficial zone of the articular cartilage,decreased chondrocytes, arrangement disorder, and duplicated tidemark; and the moderate to severe degenerative cartilagecharacterized by fissures in the deep zone of the articular cartilage, obviously decreased chondrocytes and cluster, and even fullthicknesscartilage defect. The β-catenin did not expressed in normal articular cartilage; but it expressed in the degenerativecartilage, and the expression was significantly higher in the moderate to severe degenerative cartilage than in mild degenerativecartilage (P lt; 0.05). Conclusion β-catenin plays a significant role in the pathogenesis and progression of knee primary OA,and the mechanism may be the activation of Wnt/β-catenin signaling pathway, which promotes transcri ption of inflammatorygenes and leads to the destruction of articular cartilage.