Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA 24061, USA.
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, China.
J Anim Sci. 2021 Oct 1;99(10). doi: 10.1093/jas/skab273.
Rumen epithelium plays an essential role in absorption, transport, and metabolism of short-chain fatty acids, the main products of rumen fermentation, and in preventing microbes and other potentially harmful rumen contents from entering the systemic circulation. The objective of this study was to generate an immortal rumen epithelial cell line that can be used as a convenient model of rumen epithelial cells in vitro. We isolated primary rumen epithelial cells from a steer through trypsin digestion and transduced them with lentiviruses expressing the Simian Virus (SV) 40 T antigen. We cloned the transduced cells by limiting dilution. Western blotting analysis confirmed the expression of the SV40 T antigen in two single-cell clones. Cells from one clone, named bovine rumen epithelial clone 1 (BREC1), displayed a flat and squamous morphology in culture. RNA sequencing revealed that BREC1 cells expressed many markers of epithelial cells, including keratins, the epidermal growth factor receptor, and the short-chain fatty acid transporters monocarboxylic acid transporter (MCT) 1 (MCT-1) and MCT-4. RNA sequencing revealed that BREC1 cells expressed key enzymes such as 3-hydroxymethyl-3-methylglutaryl-CoA lyase and 3-hydroxy-3-methylglutaryl-CoA synthase 1 involved in ketogenesis, a unique function of rumen epithelial cells. RNA sequencing also revealed the expression of genes encoding tight junctions, desmosomes, anchoring junctions, and polarized plasma membranes, structures typical of epithelial cells, in BREC1 cells. Cell proliferation assays indicated that BREC1 cells were similar to primary rumen epithelial cells in response to insulin-like growth factor 1, insulin, and butyrate. In conclusion, BREC1 is not only a convenient but an appropriate model for studying the factors and mechanisms that control proliferation, apoptosis, differentiation, nutrient transport, metabolism, and barrier function in rumen epithelium.
瘤胃上皮在吸收、转运和代谢短链脂肪酸（瘤胃发酵的主要产物）以及防止微生物和其他潜在有害的瘤胃内容物进入全身循环方面发挥着重要作用。本研究的目的是生成一种永生化的瘤胃上皮细胞系，可作为体外瘤胃上皮细胞的便利模型。我们通过胰蛋白酶消化从公牛中分离原代瘤胃上皮细胞，并转导表达猿猴病毒 40 大 T 抗原的慢病毒。我们通过有限稀释克隆转导细胞。Western blot 分析证实了两个单细胞克隆中 SV40 T 抗原的表达。来自一个克隆的细胞，命名为牛瘤胃上皮克隆 1（BREC1），在培养中表现出扁平的鳞状形态。RNA 测序显示 BREC1 细胞表达了许多上皮细胞标志物，包括角蛋白、表皮生长因子受体和短链脂肪酸转运蛋白单羧酸转运蛋白 1（MCT-1）和 MCT-4。RNA 测序显示 BREC1 细胞表达了酮体生成过程中的关键酶，如 3-羟甲基-3-甲基戊二酰基辅酶 A 裂解酶和 3-羟-3-甲基戊二酰基辅酶 A 合酶 1，这是瘤胃上皮细胞的独特功能。RNA 测序还显示了 BREC1 细胞中编码紧密连接、桥粒、锚定连接和极化质膜的基因的表达，这些结构是上皮细胞的典型特征。细胞增殖测定表明，BREC1 细胞对胰岛素样生长因子 1、胰岛素和丁酸盐的反应与原代瘤胃上皮细胞相似。总之，BREC1 不仅是研究控制瘤胃上皮增殖、凋亡、分化、营养转运、代谢和屏障功能的因素和机制的便利模型，也是合适的模型。
