HPAF-II，一种用于研究胰腺上皮细胞结构和功能的细胞培养模型。

HPAF-II, a cell culture model to study pancreatic epithelial cell structure and function.

作者信息

Rajasekaran Sigrid A, Gopal Jegan, Espineda Cromwell, Ryazantsev Sergey, Schneeberger Eveline E, Rajasekaran Ayyappan K

机构信息

Department of Pathology and Laboratory Medicine, UCLA David Geffen School of Medicine, Los Angeles, California 90095, USA.

出版信息

Pancreas. 2004 Oct;29(3):e77-83. doi: 10.1097/00006676-200410000-00016.

DOI:10.1097/00006676-200410000-00016

PMID:15367897

Abstract

OBJECTIVES

Epithelial cells have distinct apical and basolateral plasma membrane domains separated by tight junctions. This phenotype is essential for the directional transport functions of epithelial cells. Here we characterized a well-differentiated pancreatic epithelial cell line to establish a useful model for understanding the mechanisms involved in the regulation of junctional complexes, polarity, and disease processes in the pancreas.

METHODS

Immunofluorescence of cell junction marker proteins and electron microscopy were used to determine the presence of tight junctions, adherens junctions, and desmosomes. The functionality of tight junctions was tested by transepithelial resistance measurements and transepithelial permeability studies of nonionic molecules. Tight junction function in polarity was determined by laser scanning confocal microscopy.

RESULTS

Immunofluorescence analysis in HPAF-II cells revealed tight junction localization of ZO-1, occludin, and claudin-4; adherens junction localization of E-cadherin and beta-catenin; and desmosomal localization of desmocollin. Transmission electron microscopy showed the presence of tight junctions, adherens junctions, and des-mosomes, and freeze-fracture electron microscopy revealed the presence of distinct anastomosing tight junction strands. Transepithelial electrical resistance and permeability measurements revealed functional tight junctions. In addition, 3-dimensional images of the monolayer generated by laser scanning confocal microscopy revealed that HPAF-II cells show polarity. Immunoblotting and RT-PCR analyses revealed high expression levels of E-cadherin and Na,K-ATPase beta-subunit but low levels of the transcription factor Snail in HPAF-II cells compared with MiaPaCa-2 cells.

CONCLUSION

The HPAF-II cell line is a well-differentiated human pancreatic carcinoma cell line that should be useful as a model for studies aimed at understanding epithelial polarity, regulation of junctional complexes, and disease processes in pancreas.

摘要

目的

上皮细胞具有由紧密连接分隔的独特的顶端和基底外侧质膜结构域。这种表型对于上皮细胞的定向运输功能至关重要。在此，我们对一种高度分化的胰腺上皮细胞系进行了表征，以建立一个有用的模型，用于理解胰腺中连接复合体调节、极性和疾病过程所涉及的机制。

方法

使用细胞连接标记蛋白的免疫荧光和电子显微镜来确定紧密连接、黏着连接和桥粒的存在。通过跨上皮电阻测量和非离子分子的跨上皮通透性研究来测试紧密连接的功能。通过激光扫描共聚焦显微镜确定紧密连接在极性中的功能。

结果

HPAF-II细胞的免疫荧光分析显示，ZO-1、闭合蛋白和Claudin-4定位于紧密连接；E-钙黏蛋白和β-连环蛋白定位于黏着连接；桥粒芯蛋白定位于桥粒。透射电子显微镜显示存在紧密连接、黏着连接和桥粒，而冷冻蚀刻电子显微镜显示存在明显的吻合紧密连接链。跨上皮电阻和通透性测量显示紧密连接具有功能。此外，激光扫描共聚焦显微镜生成的单层三维图像显示HPAF-II细胞具有极性。免疫印迹和RT-PCR分析显示，与MiaPaCa-2细胞相比，HPAF-II细胞中E-钙黏蛋白和Na,K-ATP酶β亚基的表达水平较高，而转录因子Snail的水平较低。