The Construction Of PLamp2C-mRFP-EGFP-the Model To Detect The Lysosomal Membrane Permeabilization Of PC3 Cells

Objective: Utilizing the tandem GFP-m RFP( green fluorescent protein-monomeric red fluorescent protein) fluorescent reporter tagged lysosome membrane proteins to indicate the change of prostate cancer cell lysosome, constructing an in vitro screening model targeting LMP(lysosomal membrane permeabilization), which would contribute to subsequent drug screeing targeting LMP.Methods: 1. Design and build the eukaryotic expression vector of Lysosomal membrane proteins Lamp2 C which have been marked by tandem m RFP-GFP fluorescent reporter.2. Incubate cells using sphingosine in different concentration, assessing the effect on apoptosis and necrosis through flow cytometry and MTT.3. Observe the expression of plasmids which is transfected into the PC3 cells by fluorescence microscopy; Incubate PC3 cells with Lyso Tracker Blue after transfection, observing the colocalization of p Lamp2C-m RFP-EGFP and lysosomes through confocal microscope; Incubate PC3 cells transfected by p Lamp2C-m RFP-EGFP using sphingosine, assessing the change of green and red fluorescence before(after) sphingosine intervention through confocal microscope. Analyze the colocalization and the change of green and red fluorescence quantitatively by Image J.Result: 1.We built p Lamp2C-m RFP-EGFP、p Lamp2C-EGFP-m RFP、pm RFP-EGFP-Lamp2 C and p EGFP-m RFP-Lamp2 C successfully.2. Sphingosine can induce cell apoptosis.3.pLamp2C-m RFP-EGFP is the best in the four plasmids which is transfected into the PC3 cells,EGFP(enhanced green fluorescent protein) and RFP expressed normally after transient transfection of p Lamp2C-m RFP-EGFP to PC3; The Pearson ’ s correlation coefficient of colocalization between p Lamp2C-m RFP-EGFP and lysosomes was 0.8362±0.0518;Green fluorescence intensity decreased significantly(t=14.66,P﹤0.05) from(0.1190±0.0223)to(0.0639±0.0156), red fluorescence intensity changed from(0.1049±0.0101)to(0.1038±0.0109)before and after sphingosine intervention, the difference was not statistically significant(t=1.136,P﹥0.05).Conclusion: We transfect p Lamp2C-m RFP-EGFP on PC3 cells lysosomes successfully. After sphingosine intervention, green fluorescence intensity decreased significantly while red fluorescence intensity didn’t, suggesting PC3 cells lysosomal membrane permeabilization.