Whitehead R H, VanEeden P E, Noble M D, Ataliotis P, Jat P S
Ludwig Institute for Cancer Research, Melbourne Tumor Biology Branch, Australia.
Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):587-91. doi: 10.1073/pnas.90.2.587.
Intestinal mucosal cells have proved difficult to culture in vitro. Many attempts have been made to develop long-term cultures of these cells either by direct culturing or by attempting to immortalize these cells by using a range of transforming viral genes, but with little success. The recent development of a transgenic mouse bearing a temperature-sensitive mutation of the simian virus 40 large tumor antigen gene (tsA58) has enabled us to initiate conditionally immortalized cultures of epithelial cells from both small intestinal and colonic mucosa of adult mice. Crypts were isolated from either the small intestines or colons of young adult mice and cultured at the permissive temperature (33 degrees C) in medium containing conditioned medium from a human colon carcinoma cell line, LIM1863. Crypts from both tissues yielded cultures of epithelial cells that have now been in culture for more than 12 months with regular passaging. The epithelial nature of the cells has been confirmed by staining with anti-keratin antibodies. The intestinal origin of the cells was demonstrated by the ability of the cells to synthesize low levels of both brush border peptidases and a disaccharidase. The levels of expression of these enzymes were modulated by the addition of sodium butyrate or phorbol myristate acetate to the medium, which resulted in an increase in the synthesis of the peptidases and a decrease in the synthesis of the disaccharidase. The cells proliferate continuously at the permissive temperature (33 degrees C), but proliferation ceases at the nonpermissive temperature (39.5 degrees C). To our knowledge, this is the first description of the establishment of epithelial cell lines from both small intestine and colon of the same mouse strain. The success reported here indicates that this transgenic mouse will be a useful source of tissue for the study of the mechanisms that control the proliferation and eventual differentiation and senescence of the cells of the intestinal mucosa. These mice will also be a useful source of cells for attempts to culture cells from other tissues that have proved difficult to culture in vitro.
肠黏膜细胞已被证明很难在体外培养。人们进行了许多尝试，通过直接培养或使用一系列转化病毒基因使这些细胞永生化来建立这些细胞的长期培养体系，但收效甚微。最近，一只携带猿猴病毒40大肿瘤抗原基因（tsA58）温度敏感突变的转基因小鼠的培育成功，使我们能够建立成年小鼠小肠和结肠黏膜上皮细胞的条件性永生化培养体系。从小肠或成年幼鼠结肠中分离出隐窝，在含有来自人结肠癌细胞系LIM1863的条件培养基的培养基中，于允许温度（33℃）下培养。来自这两种组织的隐窝都产生了上皮细胞培养物，目前这些培养物已通过定期传代培养了超过12个月。用抗角蛋白抗体染色证实了细胞的上皮性质。细胞能够合成低水平的刷状缘肽酶和双糖酶，证明了这些细胞的肠道来源。通过向培养基中添加丁酸钠或佛波酯肉豆蔻酸酯来调节这些酶的表达水平，这导致肽酶合成增加，双糖酶合成减少。细胞在允许温度（33℃）下持续增殖，但在非允许温度（39.5℃）下增殖停止。据我们所知，这是首次报道从同一小鼠品系的小肠和结肠建立上皮细胞系。此处报道的成功表明，这种转基因小鼠将成为研究控制肠黏膜细胞增殖、最终分化和衰老机制的有用组织来源。这些小鼠也将成为尝试培养其他已证明难以在体外培养的组织细胞的有用细胞来源。
