Enwicklung neuer Vektoren mit dem caninen IL-4-Gen zur Gentherapie der Osteoarthritis beim Hund
Final Report Abstract
Canine articular chondrocytes (CAC) were isolated from knee joint of dogs euthanized for reasons not related to knee problems. Dogs under 6 years of age were selected for this study, due to the fact that the yield of chondrocytes and their regenerative capacity are lower in older dogs. The extracted CAC were adapted to two different cell culture systems i.e., monolayer (2D) and alginate based three dimensional (3D) cultures. Multiple chondrocyte marker genes were studied in both culture systems at various passages or culturing periods to monitor the cells viability, growth characteristics and morphological changes. Further an inflammation model was simulated in vitro by adding IL-1ß and TNFct to the cell culture medium to see if these cytokines could trigger a signal cascade in the cultured CAC. Similarly chondrocytes transfected with or without IL-4 constructs were also subjected to IL-1ß and T N Fa to analyse whether IL-4 could downregulate typical inflammatory mediators. The various cytokine genes and other inflammatory mediators produced in the signal cascade were quantified by quantitative real time PCR (qRT-PCR). Expression of IL-4 in CAC was monitored by ELISA and Western blot and was also quantified by qRT-PCR. To analyse the anti-inflammatory potential of canine IL-4, chondrocytes were transfected with the plasmid, pcDNA3.1-IL4 (CMV promoter), and then stimulated with IL-1ß and TNFa. A marked reduction in expression of pro-inflammatory cytokines and MMPs was observed. The expression of canine IL-4 was studied by Western blot, ELISA and qRT-PCR. For the regulatory expression of IL-4, a truncated canine COX-2 promoter of 1.2 kb was cloned into a plasmid vector substituting the CMV promoter. For the expression of IL-4 under the canine COX-2 promoter, the IL-4 gene was cloned under this promoter. The COX-2 promoter activity in vitro has been verified by a reporter assay using DsRed2 gene and was quantified by FACS. Subsequently, the responsiveness and efficiency of this construct was tested by transfecting it into CAC. Eventually, the genetically modified CAC were subjected to in vitro inflammation and the various proand anti-inflammatory mediators quantified by qRT-PCR.
