The Overexpression Of Acid Sensing Ion Channels In Articular Cartilage Cells Of AA Rats

Objective: To construct eukaryotic expression vector of acid sensing ion channel-1a(ASIC1a), then transfected it into the articular cartilage cells of AA rats to make the model of overexpression of ASIC1a. Then we observed the relative expression of the mRNA and protein of ASIC1a,detected the electrophysiology and the exudation of collagenâ…¡to discover the acid sensing ion channels'effects to the metabolism of articular cartilage cells.Methods: The rats were randomly divided into normol group and AA model group. The AA model rats were induced by CFA at left hind toes, then we observed the paw articular swelling degree of the secondary side by determining the volume meter. We also observed the morphological changes of articular cartilage by the light microscope. The culture of the articular cartilage cells of AA rats and the identification by alcian dye. Through PCR,agarose electrophoresis,recyling and purification of gelatin,to be connected with vectors,translation into competent bacteria,the collection of plasmid and the identification of double enzyme cutting to contrast the plasmid to be used for tranfection. We used lipofectamine2000 tranfection reagent to tranfect the plasmid into the articular cartilage cells,then screened out the masculine clone with G418. We also detected the relative expression of the mRNA and protein of ASIC1a by fluorescence quantitative PCR and immunocytochemistry respectively to identify whether the model of overexpression was constructed successfully, then we detected the electrophysiology and the exudation of collagenâ…¡to observed the acid sensing ion channels'effects at the level of the cells'metabolism .Results: 1,We obtained the gene of ASIC1a by the extraction and reverse transcription of the rats'brains and connected it with vectors,then we obtained enough plasmid to be used for later experiments by the colon bacillus. The plasmid was identified by enzyme cutting. The result was the purpose gene was included in the plasmid and the electrophoretic stripes were accurate and distinct.2,After the lipofectamine2000 tranfection,we had masculine clone cells steadily. After the determination of relative expression of the mRNA and protein of ASIC1a,we found that the two substances both rised relatively, so the model of overexpression was constructed successfully.3,Laser scanning confocal microscope was used to detected the internal flow of Ca²⁺, and the result was the range of the overexpression group was higher than the model group;The exudation of the collagenâ…¡of the overexpression group was more than the model group by immunohistochemistry.Conclusion: Acid sencing ion channels expressed in the articular cartilage cells of AA rats, though the intervene at the gene level to make the channels overexpressed, the Ca²⁺permeability of cell membranes changed, the exudation of collagenâ…¡also changed.These indicated that the acid sencing ion channels played a important role in the metabiolism of the articular cartilage cells of AA rats. It may provide a new target for the therapy of rheumatoid arthritis.