Tree shrews (2 newborn for cell culture and 1 adult male, weight 140±10 g at the start of the experiment) and 1 adult male Sprague-Dawley rats (for western blot analysis, weight 140±10 g at the start of the experiment) were provided by Kunming Medical University Animal Center (Kunming, China) and employed in this study. The animals were raised at 20±5°C, 40–60% humidity and a 12-h light/dark cycle with food and water ad libitum. All animal work was conducted according to the guidelines for the care and use of experimental animals established by the Ministry of Science and Technology of the People's Republic of China (approval no. 2006-398), and was approved by the ethics committee of Institute of Neuroscience, Kunming Medical University (Kunming, China). All animals were bred in separated cages.

Primary cultures of OECs were set up from newborn tree shrews and the animals were sacrificed. Olfactory bulbs were aseptically removed. The meningeal layer was stripped off with fine forceps and the tissue was enzymatically (trypsin 0.25%; 1:30 tissues to trypsin ratio) and mechanically dissociated, then incubated in 5% CO₂ at 37°C for 15 min. The cells were recovered by centrifugation (100 × g for 5 min at 37°C) in culture medium [Dulbecco's modified Eagle's medium/F12 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.)] and seeded at 1×10⁵ cells/ml into culture plates and incubated in 5% CO₂ at 37°C. Cells were cultured for 10 h and then the supernatant was removed to another culture plate.

Antibodies against p75 neurotrophin receptor (p75NGFR) and BrdU were used to study the purity and proliferation of OECs. For each antibody, a minimum of two whole series of different animals were studied. Western blot analysis with protein extracts from whole tree shrew and rat brains ensured specificity of the antibodies used. The tissues were homogenized in RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) that contained a mixture of proteinase inhibitors and the supernatant was collected by centrifuging at 4°C at 22,500 × g for 15 min. The protein concentration was determined by the DC protein assay from Bio-Rad Laboratories, Inc. (Hercules, CA, USA), and protein samples (100 µg) of each cell lysate in SDS loading buffer (Biosharp) were electrophoresed on 10% SDS-PAGE gel. Electrophoresis was performed and the proteins were transferred onto polyvinylidene difluoride membranes (Pall Life Sciences, Port Washington, NY, USA) using electroblotting apparatus (Bio-Rad Laboratories, Inc.). The membranes were blocked at 37°C for 1 h in TBS containing 0.1% Tween-20 and 5% dried milk, and were then incubated at 4°C overnight with NGFRp75 antibody (1:1,000; cat. no. AB1554; EMD Millipore, Billerica, MA, USA) They were incubated with the IRDye 800-conjugated affinity purified goat anti-rabbit immunoglobulin (Ig)G (1:5,000; cat. no. 611-1302; Rockland Immunochemicals, Inc., Pottstown, PA, USA) for 1 h at room temperature and visualization using an Odyssey laser scanning system (LI-COR Biosciences, Lincoln, NE, USA). Blots were reprobed with the monoclonal mouse anti-β actin antibody (1:5,000; cat. no. A2228; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), followed by reaction with IRDye 800-conjugated affinity purified goat anti-mouse IgG (1:5,000; cat. no. 610-1302; Rockland Immunochemicals, Inc.) at 37°C for 2 h to confirm equal protein loading.

Immunofluorescence was performed on OECs. The cells were fixed with 4% formalin solution at room temperature for 5 min then washed with PBS and permeabilized with 0.1% Triton X-100. The cells were then blocked with 1% bovine serum albumin (10082-139; Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 30 min and incubated for 18 h at 4°C with anti-p75NGFR (1:500; AB1554; EMD Millipore) and anti-BrdU (1:200; mAb #5292; Cell Signaling Technology, Inc., Danvers, MA, USA) antibodies. Following washing with PBS, the cells were incubated at 37°C for 2 h with respective anti-Mouse IgG H&L (Alexa Fluor 647; 1:500; ab150115; Abcam, Cambridge, MA, USA). Following incubation, the slides were washed with PBS, mounted and examined under a fluorescence microscope (Leica TCS SP2; Leica Microsystems GmbH, Wetzlar, Germany). Fluoroshield mounting medium with DAPI (Sigma-Aldrich; Merck KGaA) was used for DAPI staining and the staining was performed according to the manufacturer's protocol.

Total RNA from OECs was quantified using a NanoDrop ND-1000 (Thermo Fisher Scientific, Inc.) and RNA integrity was assessed using standard denaturing agarose gel electrophoresis (27) (performed by KangChen Biotech Co., Ltd., Shanghai, China). Total RNA from each sample was used for labeling and array hybridization as follows: i) Reverse transcription using the Superscript ds-cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.); ii) ds-cDNA labeling using the NimbleGen One-Color DNA Labeling kit (Roche NimbleGen, Inc., Madison, WI, USA); iii) array hybridization using the NimbleGen Hybridization System, followed by washing with the NimbleGen wash buffer kit (Roche NimbleGen, Inc.); and iv) array scanning using the Axon GenePix 4000B microarray scanner (Molecular Devices, LLC, Sunnyvale, CA, USA). Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5; Roche NimbleGen, Inc.) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. The Probe level (*_norm_RMA.pair) files and Gene level (*_RMA.calls) files were generated following normalization. All gene level files were imported into Agilent GeneSpring GX software (version 11.5; Agilent Technologies, Inc., Santa Clara, CA, USA) for further analysis. Finally, hierarchical clustering was performed using Gene Ontology (http://www.geneontology.org) to show distinguishable gene expression profiling among samples.

Western blotting with protein extracts from whole tree shrew and rat brains ensured specificity of the antibodies used in the present study. Western blotting demonstrated that the antibodies produced bands that were located at the same level in the gel for rat and tree shrew brain protein extracts. Notably, the western blots also demonstrated that these bands had the proper molecular weight for the p75NGFR antibody examined (Fig. 1A).

To investigate the method of culturing OECs from tree shrews, the bulbus olfactorius of newborn tree shrews was used. Cultured for 24 h, certain cells began to demonstrate adherence and assume a round or star-like shape (Fig. 1B1). Then, 6 days following adherent culture, three classic morphologies of OECs were observed: Bipolar or fusiform and oblate (Fig. 1B2). Among these shapes, fusiform and oblate were more common, and an evident three-dimensional appearance. After 14 days, the number of cells did not significantly increase, although the cells were still growing rapidly (data not shown). Cells were stained against BrdU (Fig. 1B3) to confirm their capacity for proliferation, which demonstrated that all stages of OECs nuclei were positively stained, demonstrating that they possessed proliferative ability. Immunocytochemical staining against p75NGFR (Fig. 1B4) demonstrated that the fusiform cells or their nuclei were positively stained, demonstrating that they were OECs. All cell nuclei were positively stained with DAPI (Fig. 1B5) and the merged image is demonstrated in Fig. 1B6.

In order to investigate RNA integrity and genomic DNA contamination, the denaturing agarose gel electrophoresis test was used. The result demonstrated that optical density (OD) A260/A280 ratio was 2.00 and the OD A260/A230 ratio was 2.35. These results confirmed that the RNA was pure [for spectrophotometer, the O.D. A260/A280 ratio should be close to 2.0 for pure RNA (ratios between 1.8 and 2.1 are acceptable) The OD A260/A230 ratio should be >1.8]. The 28S and 18S ribosomal RNA bands were fairly sharp, intense bands and there was a diffuse smear of ethidium bromide-stained material migrating between the 18S and 28S ribosomal bands, possibly comprising mRNA and other heterogeneous RNA species (Fig. 2A). Prior to analysis a boxplot was performed (Fig. 2B). Then, gene chip assay suggested that OECs expressed 9,821 genes (Fig. 2C) and 45 important genes are listed in Table I and Fig. 2D and E.