Haslam I S, Simmons N L
Stopford Building, University of Manchester, Manchester, M13 9PT, UK.
J Comp Physiol B. 2014 Jul;184(5):673-81. doi: 10.1007/s00360-014-0804-5. Epub 2014 Apr 20.
Rumen fermentation of plant-based forage in bovines is the major site for generation and absorption of short-chain fatty acids. Consequentially, the rumen is also the site for initial exposure to toxins released from diet. Accordingly, we have investigated the expression of bovine ABC transporters in the rumen associated with cytoprotection against xenobiotic exposure, namely MDR1 (ABCB1), MRP2 (ABCC2) and BCRP (ABCG2). Bovine rumen samples from the ventral sac were obtained post-mortem from a commercial slaughterhouse after humane killing. Rumen papilla samples were then prepared for total RNA isolation for RT-PCR, SDS-PAGE/Western blotting and immunohistochemistry. PCR products of the predicted size were observed for both MDR1 and BCRP, but not for MRP2 using bovine-specific primers. β-actin was used as a control transcript. Western blot analysis using C219 primary monoclonal antibody revealed MDR1 protein expression in bovine rumen (Mapp, of ~170-180 kD). Immunolocalisation of MDR1 using UIC2 monoclonal antibody within cryosections of bovine rumen showed extensive membrane staining in the cells of the stratum granulosum, stratum spinosum and stratum basale. MDR1 expression was absent from outer stratum corneum. Protein expression and immunolocalisation were also confirmed for BCRP, with prevalent staining in the stratum basale, becoming weaker in the stratum spinosum and stratum granulosum.
牛体内基于植物的草料在瘤胃中的发酵是短链脂肪酸生成和吸收的主要部位。因此，瘤胃也是最初接触日粮释放毒素的部位。据此，我们研究了牛瘤胃中与针对异源生物暴露的细胞保护相关的ABC转运蛋白的表达，即多药耐药蛋白1（MDR1，ABCB1）、多药耐药相关蛋白2（MRP2，ABCC2）和乳腺癌耐药蛋白（BCRP，ABCG2）。在人道屠宰后，从一家商业屠宰场获取牛腹囊的瘤胃样本。然后制备瘤胃乳头样本用于提取总RNA，以进行逆转录聚合酶链反应（RT-PCR）、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳/蛋白质免疫印迹（SDS-PAGE/Western blotting）和免疫组织化学分析。使用牛特异性引物观察到MDR1和BCRP出现了预测大小的聚合酶链反应产物，但未观察到MRP2的产物。β-肌动蛋白用作对照转录本。使用C219原发性单克隆抗体进行的蛋白质免疫印迹分析显示牛瘤胃中有MDR1蛋白表达（分子量约为170 - 180 kDa）。在牛瘤胃冰冻切片中使用UIC2单克隆抗体对MDR1进行免疫定位显示，在颗粒层、棘层和基底层细胞中有广泛的膜染色。角质层外层未检测到MDR1表达。也证实了BCRP的蛋白表达和免疫定位，其在基底层普遍染色，在棘层和颗粒层中染色变弱。
