Abstract
Objective Utilizing the tandem green fluorescent protein (GFP)-monomeric red fluorescent protein (mRFP) fluorescent reporter tagged lysosome membrane proteins to indicate the change of prostate cancer cell lysosome, constructing an in vitro screening model targeting lysosomal membrane permeabilization(LMP), which would contribute to subsequent drug screeing targeting LMP. Methods (1) Design and build the eukaryotic expression vector of Lysosomal membrane proteins Lamp2C which have been marked by tandem mRFP-GFP fluorescent reporter. (2) Observe the expression of pLamp2C-mRFP-EGFP which is transfected into the PC3 cells by fluorescence microscopy. (3) Incubate PC3 cells with LysoTracker Blue concentration in 55 nmol/L medium after transfection, observing the colocalization of pLamp2C-mRFP-EGFP and lysosomes through confocal microscope. (4) Incubate PC3 cells transfected by pLamp2C-mRFP-EGFP using sphingosine concentration in 40 μmol/L, 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. Results (1) We built pLamp2C-mRFP-EGFP successfully. (2) enhanced green fluorescent protein(EGFP) and RFP expressed normally after transient transfection of pLamp2C-mRFP-EGFP to PC3. (3) The Pearson's correlation coefficient of colocalization between pLamp2C-mRFP-EGFP and lysosomes was 0.836 2±0.051 8. (4) Green fluorescence intensity decreased significantly (t=14.66, P 0.05). Conclusion We transfect pLamp2C-mRFP-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. Key words: Lysosomal membrane permeabilization; Tander monomeric red fluorescent protein-green fluorescent protein fluorescent reporter; Prostate cancer cell; Sphingosine; Apoptosis
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