Jan 25, 1997 - mented with 10% FCS (Freshney 1986). After reaching con- fluence, the cells were subcultured. Cells at the 7â10th passages were used for the ...
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Effect of water-soluble extract from antler of wapiti (Cervus elaphus) on the growth of fibroblasts H. H. Sunwoo, T. Nakano, and J. S. Sim1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5. Received 8 November 1996, accepted 25 January 1997. Sunwoo, H. H., Nakano, T. and Sim, J. S. 1997. Effect of water-soluble extract from antler of wapiti (Cervus elaphus) on the growth of fibroblasts. Can. J. Anim. Sci. 77: 343–345. The water-soluble extracts were prepared from the tip sections of antlers of 4-yr-old wapiti stags, and the effect of extract on the growth of bovine skin fibroblasts in culture was examined. The results showed the presence of growth promoting factor(s) in the antler extract. The stimulation of cell growth was found to be dose dependent (P < 0.05). Key words: Growth factor, antler, wapiti, fibroblast Sunwoo, H. H., Nakano, T. et Sim, S. 1997. Effets d’extraits hydrosolubles des bois du wapiti (Cervus elaphus) sur la croissance des fibroblastes. Can. J. Anim. Sci. 77: 343–345. Des extraits hydrosolubles obtenus à partir des extrémités des bois de cerfs d’Amérique de quatre ans ont été utilisés sur des fibroblastes de la peau de bovins. Les résultats révèlent la présence dans les extraits de facteurs de croissance dont l’activité stimulatrice sur la croissance cellulaire étaient liés à la dose d’utilisation (P < 0,05). Mots clés: Facteur de croissance, bois, wapiti, fibroblaste
Antlers are unique mammalian structures, in which a cycle of growth, maturation, mineralization, casting and regeneration occurs annually. Antlers are probably the fastest growing non-pathological tissues. For example, the maximum growth rate of antlers of elk (Cervus elaphus) is approximately 2.75 cm d–1 (Goss 1970). There is, however, limited information concerning growth factors involved in the development of antler. Suttie et al. (1991) analyzed growth factors in the blood of red deer and reported a close association of insulin growth factor I with antler growth. Ko et al. (1986) reported the presence of EGF in the velvet tissue from the growing antler of red deer, and partially characterized it. This report describes our preliminary study using a cell culture system to monitor growth-promoting activity in the water-soluble extract from the growing antlers of wapiti. The authors, however, did not determine whether the EGF was derived from the velvet skin or the inner tissue containing the growth center of antler. Fresh samples of velvet antlers were obtained in June at 65 d after button-casting from four 4-yr-old wapiti stags averaging 365 kg body weight. These animals were kept at the University of Alberta Ministik Research Station. Care of animals and procedures used were in accordance with the guidelines of the Canadian Council on Animal Care (1993). The sample of tip section containing the growth center of antler (Banks 1974) was taken from the main beam of each antler, washed in cold water, dissected free of velvet skin and stored at –20°C until extracted. 1Author
To obtain the water-soluble extract, frozen samples of tip sections of antlers were thawed at 4°C, and homogenized with cold (4°C) deionized water (5 mL 100 mg–1 sample). The extracts were centrifuged at 40 000 × g, and 4°C for 30 min, and the supernatant used for cell culture study was sterilized through 0.22-µm filters (Millipore Corporation, Bedford, MA). The protein contents in the water-soluble extract were determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Gel chromatography of water-soluble extract was carried out at 4°C using a 1.0 × 110-cm column of Sephacryl S-300 (Pharmacia Biotech Canada Inc. Baie d’Urfe, QC), which was equilibrated and eluted with PBS (0.14 M NaCl, 0.0027 M KCl, 0.0081 M Na2HPO4, and 0.0015M KH2PO4). Fractions (1 mL) collected at a flow rate of 3 mL h–1 were monitored for absorbance at 280 nm to determine protein contents. Appropriate fractions obtained were pooled, filtered through 0.22-µm membrane and used for cell culture study (see below). Bovine skin fibroblasts were isolated from the fresh skin from young adult cattle, obtained at a local abattoir (Gainers Inc., Edmonton, AB), and were cultured in DMEM supplemented with 10% FCS (Freshney 1986). After reaching confluence, the cells were subcultured. Cells at the 7–10th passages were used for the cell culture experiment. These cells were seeded at a density of 30 000 cells in 2 mL of Abbreviations: DMEM, Dulbecco’s Modified Eagles Medium; EGF, epidermal growth factor; FCS, fetal calf serum; PBS, phosphate-buffered saline
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Fig. 1. Effect of the water-soluble extract from wapiti antler on the growth of bovine skin fibroblasts. Bovine skin fibroblasts were cultured, in triplicate, in the medium containing different amounts of the water-soluble extract of antler (0, 1.5, 3, 4.5 and 6 mg as protein), and the growth of fibroblasts was examined by counting cell numbers through a hemocytometer. See the text for details. Each bar represents the mean (± standard error of mean) from three separate experiments. The increase in cell number was dose dependent (P < 0.05).
DMEM supplemented with 10% FCS in six-well Costar plastic culture plates (Costar Corporation, Cambridge, MA), and incubated in 5% CO2 at 37°C. The medium was replaced 24 h after seeding with 2 mL of DMEM with 2% FCS. Preliminary experiments with different levels (0, 2, 5 and 10%) of FCS showed suboptimal cell growth (see below) with 2% FCS, which was considered to be the appropriate condition to detect the effects of water-soluble extract and its fractions on cell growth. The increase in number of cells with 2% FCS was slower than that with 5 or 10% FCS, and reached a plateau after 3 d of incubation. In contrast, the number of cells continued to increase with 5 and 10% FCS, in that the rate of increase was higher with the latter. No appreciable growth of bovine skin fibroblasts was observed without FCS. After replacement by 2% FCS, cells were incubated with different amounts of water-soluble extract in 5% CO2 at 37°C for 72 h to determine the effect of extract on cell growth. Samples of 1.5 to 6 mg of extract, measured as protein, in 100 µL of PBS were added to 2 mL of medium. For the experiment to examine the mitogenic effect of fractions obtained from a Sephacryl S-300 column, samples of 20 µg protein in 100 µL PBS were added to 2 mL of medium. In both experiments, cell culture with no added water-soluble extract (or its fraction) was used as control. Cell numbers were counted with hemocytometer after trypsinization with
Fig. 2. Sephacryl S-300 (1 × 110 cm) chromatograms of the watersoluble extract from wapiti antler. Fractions in each of five protein peaks (I–V) obtained were pooled for further study. Void volume (Vo) and total volume (Vt) of the column were determined using blue dextran (Pharmacia Biotech. Canada Inc.) and tritiated water, respectively.
0.25% trypsin for 3 min at 37°C. A t-test was used to detect significant differences (P < 0.05) between means of cell culture. The response of differenc concentrations of water-soluble extract on the growth of bovine skin fibroblasts is shown in Fig. 1. Addition of water-soluble extract promoted (P < 0.05) the growth of fibroblasts. The increase of cell number was dose-dependent suggesting the presence of growth-promoting activity in the antler extract. The water-soluble extract was then fractionated using gel chromatography on Sephacryl S-300 to examine which fraction had growth-promoting activity in the cell culture system. The elution pattern for protein is shown in Fig. 2. Five protein peaks, I (Kav, 0), II (Kav, 0.20), III (Kav, 0.33), IV (Kav, 0.74), and V (Kav, 0.84) were obtained, which accounted for 14.2, 20.1, 45.8, 10.0 and 9.9% of total protein recovered, respectively. The protein peak fractions were pooled as indicated in Fig. 2, and tested for a mitogenic activity in fibroblast culture. Only the pooled material from peak I, which was excluded from the column, demonstrated growth-promoting effect (P < 0.05) (Fig. 3). Further characterization of the material was not pursued in this study. Ko et al. (1986) reported the presence of EGF in the water-soluble extract from red deer antler. These authors chromatographed the extract on a column of Sephadex G50, and obtained two peaks of EGF detected by radioimmunoassay, one of which eluted at the void volume, and a second which was retarded in the column. Since the exclusion limit of Sephadex G-50 is 50 times lower than that of Sephacryl S-300 (supplier’s information), it is possible that the size of EGF retarded in the Sephadex G-50 is much smaller than that of peak I fraction excluded from Sephacryl S-300 (Fig. 2). It remains to be determined if the peak I fraction from wapiti antler contains EGF and/or growth factor(s) other than EGF.
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produce the peak I material with fibroblast growth promoting activity. In conclusion, the present results indicate that the tip section of wapiti antler without velvet skin contains growth factor(s), which can stimulate the growth of bovine fibroblast in culture.
Fig. 3. Effects of fractions of water-soluble extract on the growth of bovine fibroblasts. *Mean greater than that of control culture with PBS.
The tip section of wapiti antler consists of zones of reserve mesenchymes, prechondroblasts, chondroblasts, and chondrocytes (H. H. Sunwoo and T. Nakano, unpublished observations). It is interesting to know which types of cells
Banks, W. J. 1974. The ossification process of the developing antler in the white-tailed deer (Odocoileus virgianus). Calc. Tiss. Res. 14: 257–274. Canadian Council on Animal Care. 1993. Guide to the care and use of experimental animals. 2nd ed. Vol. 1. E. D. Olfert, B. M. Cross, and A. A. McWilliam, eds. CCAC, Ottawa, ON. Freshney, R. I. 1986. Animal cell culture-a practical approach. R. I. Freshney, ed. IRL Press, Oxford, UK. Goss, R. J. 1970. Problems of antlerogenesis. Clin. Orthop. 69: 227–238. Ko, K. M., Tsao, S. W., Yip, T. T., Kong, Y. C., Fennessy, P., Belew, M. C. and Porath, J. 1986. Epidermal growth factor from deer (Cervus elaphus) submaxillary gland and velvet antler. General Comp. Endocrinol. 63: 431–440. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265–275. Suttie, J. M., Corson, I. D., Gluckman, P. D. and Fennessy, P. F. 1991. Insulin like growth factor-I, growth and body composition in red deer stags. Anim. Prod. 53: 237–242.
