Tashima Kimihito, Zhang Songhua, Ragasa Regina, Nakamura Eiji, Seo Ji Hye, Muvaffak Asli, Hagen Susan J
Department of Surgery, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
Am J Physiol Gastrointest Liver Physiol. 2009 Feb;296(2):G319-29. doi: 10.1152/ajpgi.90355.2008. Epub 2008 Nov 20.
The physiology of gastric epithelial cells is often studied by using cancer cell lines, which may or may not provide information relevant to normal cells. Because few models exist to study chief cell physiology in vitro, our purpose was to develop primary cultured chief cells from rodent species that are structurally and functionally similar to native chief cells. For this, isolated chief cells from the rat stomach, purified by counterflow elutriation and density gradient centrifugation, were grown in media with growth factors. Purity and the continuity of tight junctions were determined, and permeability, viability, transepithelial resistance (TER), cell number and proliferation, and pepsinogen secretion in response to carbachol were measured. When plated in media alone or with basic fibroblast growth factor, the isolated chief cells attached by 2 days and were confluent by 4 days after seeding. However, tight junctions were discontinuous, TER was less than 300 Omega cm(2), and permeability was high. In contrast, chief cells incubated with hepatocyte growth factor (HGF) were confluent in 3 days and had a TER greater than 2,000 Omega cm(2), continuous tight junctions, and low permeability. EGF was intermediate. HGF facilitated monolayer development by increasing cell number, which occurred by the proliferation of chief cells. Chief cell cultures, grown with HGF, consisted of more than 99% gastric intrinsic factor-expressing cells and showed robust pepsinogen secretion. Coexpression studies for neck and chief cell markers suggest that the cultures are a mixture of mature, immature, and transitional zone cells. This model will be useful for investigating mechanisms that regulate chief cell physiology in health and disease.
胃上皮细胞的生理学研究通常使用癌细胞系，这些细胞系可能提供也可能不提供与正常细胞相关的信息。由于体外研究主细胞生理学的模型很少，我们的目的是从啮齿动物物种中培养出在结构和功能上与天然主细胞相似的原代主细胞。为此，从大鼠胃中分离出的主细胞，通过逆流淘析和密度梯度离心纯化后，在含有生长因子的培养基中培养。测定了纯度和紧密连接的连续性，并测量了通透性、活力、跨上皮电阻（TER）、细胞数量和增殖以及对卡巴胆碱的胃蛋白酶原分泌。当单独接种于培养基或与碱性成纤维细胞生长因子一起接种时，分离出的主细胞在接种后2天附着，4天汇合。然而，紧密连接是不连续的，TER小于300Ω·cm²，通透性很高。相比之下，用肝细胞生长因子（HGF）孵育的主细胞在3天内汇合，TER大于2000Ω·cm²，紧密连接连续，通透性低。表皮生长因子（EGF）的作用介于两者之间。HGF通过增加细胞数量促进单层发育，这是由主细胞增殖引起的。用HGF培养的主细胞培养物由超过99%表达胃内因子的细胞组成，并显示出强大的胃蛋白酶原分泌。颈部和主细胞标志物的共表达研究表明，这些培养物是成熟、未成熟和过渡区细胞的混合物。该模型将有助于研究健康和疾病状态下调节主细胞生理学的机制。
