Establishment of an immortalized intestinal epithelial cell line from tree shrews by lentivirus-mediated hTERT gene transduction

Bowen Yin,Xuan Wang,Xiaofei Li,Xiaomei Sun,Yuanyuan Han,Lingxia Chen,Qingkai Song,Jiejie Dai

doi:10.1007/s10616-018-0270-0

Bowen Yin, Xuan Wang + Show 6 more

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https://doi.org/10.1007/s10616-018-0270-0

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Abstract

The intestinal epithelium has an average lifespan of 4–5 days. Normally, primary intestinal epithelial cells can be cultured for about 15 days in vitro. The aim of this study was to explore methods to isolate and immortalize intestinal epithelial cells (IECs) of tree shrews in order to establish a new resource of experimental material and to provide a cell model for drug development and infection mechanism research. Tissue from the small intestine of tree shrews was digested with collagenase XI, neutral protease I, and dithiothreitol. The human telomerase reverse transcriptase gene (hTERT) was transferred into tree shrew IECs using the pHBLV-CMVIE-ZsGreen-Puro vector. The level of hTERT mRNA was detected by quantitative reverse transcription polymerase chain reaction. Immunofluorescence and western blot assays were performed to detect biochemical markers of IECs. The micromorphology of cells was observed with electron microscopy. We then conducted experiments to assess proliferative activity and analyze the karyotype of isolated cells. The results showed the immortalized cell line that we established and screened, maintained the characteristics and biochemical markers of primary IECs. Our results showed that the cell line we established can be considered an alternative cell model for intestinal drug research and for studies on intestinal infection and cell signaling.

Highlights

Tree shrews (Tupaia belangeri chinesis) are small mammals that are close relatives of primates and show a high degree of similarity to humans in their anatomy and physiology, neural development, and responses to viral infection and psychological stress (Xu et al 2013)
Recent studies have revealed that the intestinal microenvironment is complex and that dysbiosis can result in various diseases (Tanaka and Nakayama 2017), including inflammatory bowel disease (Christopher et al 2015), irritable bowel syndrome (Codling et al 2010), obesity (Karimi et al 2015), allergies (Lynch and Boushey 2016), autoimmune diseases, and brain disorders (Wang and Kasper 2014)
The remaining tissue was placed in 10 mL of digestion buffer containing 1% v/v fetal bovine serum (FBS), 75 U/ mL collagenase XI, 20 lg/mL dispase neutral protease II, and 0.5 mM DTT in Dulbecco’s Modified Eagle’s Medium (DMEM)

Summary

Introduction

Tree shrews (Tupaia belangeri chinesis) are small mammals that are close relatives of primates and show a high degree of similarity to humans in their anatomy and physiology, neural development, and responses to viral infection and psychological stress (Xu et al 2013). Compared with other non-primate mammals, tree shrews have a high brain-to-body mass ratio (Xu et al 2012). Other characteristics, such as their small size, short breeding cycle, and low food and research costs make the tree shrew an ideal animal model. The intestinal epithelium forms a natural physical barrier and maintains the structural stability of the intestine. Models of the intestinal epithelium are important for understanding these diseases. Long-term in vitro culture of primary intestinal epithelial cells (IECs) is difficult because the epithelium is completely renewed every 4–5 days (van der Flier and Clevers 2009). IECs are highly sensitive to anoikis, a form of programmed cell death caused by epithelial cell and extracellular matrix disorders.

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