Expression Of Matrix Metalloproteinase-3(MMP-3) In Lens Epithelial Cells

ObjectiveThe aim of this study is to construct eukaryotic recombination plasmid andexplore the possibility of the success of constructing an eukaryotic recombinationplasmid with pEGFP-N1and MMP-3. Way through the primary cultured pig lensepithelial cells cultured. The eukaryotic recombination plasmid pEGFP-N1-MMP-3was transfected into LECs and the expression products was detected by Western blot.The expression level of MMP-3was observed in order to provided theoretical basisfor the research of the expression and the function of the protein in LECs.Methods1. The biological company was entrust with the synthesis of human MMP-3.Bacterial strain contain pEGFP-N1and another bacterial strain contain MMP-3wereinoculated on LB medium for37℃. After12-16hours, target plasmid was extracted.The plasmid MMP-3was digested by XHOⅠ and BglⅡ and the vector pEGFP-N1was digested by EcoRⅠ and BglⅡ. Target fragment from the products of enzymedigestion were treated by gel recovered. Then target fragment were connected and therecombination plasmid was amplified. The correctness of recombination plasmid wasidentified by restriction endonuclease digesting and DNA sequencing.2. Lens were taken out from the eyes of pig and were washed by PBS whichcontains antibiotics. Lens capsular was torn out carefully under operation microscope.The capsular was digested by0.25%pancreatic enzyme, then the supernatant wassucked away and was shift in DMEM medium which contain10%fetal calf serum toterminate the digestion. After centrifugating the solution, the supernatant was takenaway. The raffinate was shift into culture dish to cultivate by cell culture system.LECs were observed continually under fluorescence microscope. At the same time,cell picture were collected. The eukaryotic recombination plasmid was transfected into LECs. It compare with control group that lipidosome without recombinationplasmid was transfected into LECs. After48hours, the pictures were collected underfluorescence microscope. Gathering cells transfected and preparing the protein sample.The expression level of fibronectin was detected by equipment. The sample level wascalculated. After SDS-PAGE, the expression level of sample protein was detectedby western blot. Finally, the result of western blot was analyzed by software.Results1. Construction of the eukaryotic recombination plasmid（1）The vector pEGFP-N1was digested by restriction endonuclease EcoRⅠand BglⅡ, and the electrophoresis of the digested products showed one fragment:4700bp fragment. The result conforms to the length of pEGFP-N1. And the syntheticplasmid MMP-3was digested by restriction endonuclease XHOⅠ and BglⅡ, andthe electrophoresis of the digested products showed two fragment:2700bp fragmentand1400bp fragment. These results respectively conform to the length of the vectorpMD18-T and the length of target fragment MMP-3.（2） The electrophoresis of the eukaryotic recombination plasmidpEGFP-N1-MMP-3showed that one fragment, and the consistency of therecombination plasmid was so thick that it could be transfected. Then it was digestedby EcoRⅠ and BglⅡ, and the electrophoresis of the digested products showed twofragment:4700bp fragment and1400bp fragment. Two fragment respectivelyrepresent pEGFP-N1and MMP-3.（3）The result of DNA sequencing analysis was consistent with the sequencepublished in GeneBank. And the similarity was99.6%.2. Culture and transfection of the primary cell（1）After3-5days, diffuse cell group were found under microscope. About10days, cells were confluence gradually under microscope. About two weeks, allcells were confluence.（2）The expression of MMP-3in LECs: control group: under microscope,cells arrange regularly; they were achromic and there is no GFP fluorescence under fluorescence microscope. Transfection group: cells were polygonal and irregular.They were achromic as well. Under fluorescence microscope, there were some cellsthat were transfected successfully and cells were GFP fluorescence.（3）We have analyzed the result of western blot by software and found thatthe expression of fibronectin in cells of control group was more than transfectiongroup.（4）Compared with control group, the expression level of fibronectin ofcontrol group cells was reduced.Conclusion1. Eukaryotic recombination plasmid can be construct successfully with targetfragment MMP-3and carrier pEGFP-N1.2. Cells were active in the process of LECs primary cultured. After theeukaryotic recombination plasmid was transfected into LECs, the cells can expressMMP-3protein. The difference of the expression of the fibronectin between controlgroup and transfection group is evident and the expression level of the former morethan the latter. One of the function of MMP-3is to degrade ECM such as fibronectin.Then it further confirms the successful expression of MMP-3protein and thephysiological function.