Effects of coxsackie-adenovirus′receptor siRNA on the permeability of Sertoli cell epithelial barrier and its related mechanism

LI Xing-wang; ZHAO Yan-ling

doi:10.3969/j.issn.1002-266X.2014.35.001

Abstract：Objective To investigate the effect of coxsackie-adenovirus receptor(CAR) siRNA on the permeability of Sertoli cell epithelial barrier and its related mechanism.Methods By using two-compartment primary culture system, Sertoli cell epithelial barrier was established.After 3 days of culture, the cells were divided into control and CAR siRNA groups, in which the cells were transfected with nontargeting or CAR-specific small interfering RNA duplexes, respectively.2 d after the end of transfection, the expression of CAR mRNA and protein was measured by TR-PCR and Western blotting, and the transepithelial electrical resistance (TER) was assessed using Millicell-ERS electrical resistance system.The distribution of occludin was observed with fluorescence microscope, the total amount of occludin and the amount of occludin combined with early endosome antigen 1 ( EEA-1) was examined by Western blotting.Results The mRNA and protein expression levels of CAR in CAR siRNA group (0.122 ±0.013 and 0.142 ±0.041) were significantly lower than those in control group (0.429 ±0.039 and 0.532 ±0.022), all P<0.05.TER in CAR siRNA group was (37 ±2.5)ohm· cm2, which was signicantly lower than that in control group(50 ±3.0) ohm· cm2 , P<0.05.There was no signcant difference in protein expression of oc-cludin between siRNA group and control group (0.164 ±0.025, 0.143 ±0.031) , P>0.05.In control group, occludin was localized at the edge of cell, and the pattern of its distribution was beehive-like and line-like.However, the distribution of occludin in cells in CAR siRNA group was interrupted, and it moved from the edge of cells to cytoplasm.The amount of oc-cludin combined with EEA1 in CAR siRNA group (1.332 ±0.018) was significantly higher than that in control group (1.00 ±0.015), P<0.05.Conclusion CAR siRNA can increase the permeability of Sertoli cell epithelial barrier, and its mech-anism may be associated with maldistribution of occludin induced by the increment in endocytosis.