CAR siRNA对睾丸支持细胞上皮屏障通透性的影响及机制

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

目的观察柯萨奇病毒—腺病毒受体(CAR)siRNA对睾丸支持细胞上皮屏障通透性的影响,并探讨其机制。方法采用睾丸支持细胞原代双室培养方法制备睾丸支持细胞上皮屏障,细胞培养3 d后分为CAR siRNA组、对照组,分别转染CAR siRNA、无同源性非靶向双链RNA。转染后第2天,分别采用RT-PCR和Western blotting法检测睾丸支持细胞中CAR mRNA、蛋白表达;采用Millicell-ERS电阻系统测量双室模型内外室的电位差;采用免疫荧光细胞化学染色法检测支持细胞的Occludin蛋白,荧光显微镜下观察Occludin分布情况;采用Western blotting法检测支持细胞中总Occludin蛋白量、与早期内涵体抗原1(EEA1)抗体结合的Occludin蛋白。结果 CAR siRNA组与对照组CAR mRNA相对表达量分别为0.122±0.013、0.429±0.039,CAR蛋白相对表达量分别为0.142±0.041、0.532±0.022,两组比较,P均<0.05。CAR siRNA组与对照组TER分别为(37±2.5)、(50±3.0)ohm·cm2,两组比较,P<0.05。CAR siRNA组与对照组Occludin蛋白相对表达量分别为0.164±0.025、0.143±0.031,两组比较,P>0.05。对照组Occludin呈蜂巢样沿细胞膜线状分布;CAR siRNA组Occludin分布较紊乱,细胞膜处减少,线性分布破坏,细胞质内增多。CAR siRNA组、对照组细胞中与EEA1结合的Occludin蛋白量分别为1.332±0.018、1.000±0.015,两组比较,P<0.05。结论 CAR siRNA能增加睾丸支持细胞上皮屏障通透性,其机制可能与其诱导Occludin蛋白内吞增强而分布改变有关。

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＆lt;0.05.TER in CAR siRNA group was （37 ±2.5）ohm＆#183; cm2, which was signicantly lower than that in control group（50 ±3.0） ohm＆#183; cm2 , P＆lt;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＆gt;0.05.In control group, occludin was＆amp;nbsp;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＆lt;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.