ESTABLISHMENT OF CONDITIONALLY IMMORTALIZED EPITHELIAL-CELL LINES FROM BOTH COLON AND SMALL-INTESTINE OF ADULT H-2KB-TSA58 TRANSGENIC MICE
ESTABLISHMENT OF CONDITIONALLY IMMORTALIZED EPITHELIAL-CELL LINES FROM BOTH COLON AND SMALL-INTESTINE OF ADULT H-2KB-TSA58 TRANSGENIC MICE
复制标题
DOI：
10.1073/pnas.90.2.587
复制
发表时间：
1993-01-15
影响因子：
11.1
通讯作者：
JAT, PS
中科院分区：
文献类型：
--
作者：
WHITEHEAD, RH;VANEEDEN, PE;JAT, PS
肠粘膜细胞已被证明难以在体外培养。已经进行了许多尝试来开发这些细胞的长期培养物，通过直接培养或通过使用一系列转化病毒基因试图使这些细胞永生化，但几乎没有成功。最近开发的转基因小鼠携带的猿猴病毒40大肿瘤抗原基因（tsA58）的温度敏感性突变，使我们能够启动有条件的永生化培养的上皮细胞从小肠和结肠粘膜的成年小鼠。从年轻成年小鼠的小肠或结肠中分离隐窝，并在允许温度（33 ℃）下在含有来自人结肠癌细胞系LIM1863的条件培养基的培养基中培养。来自两种组织的隐窝产生上皮细胞的培养物，这些上皮细胞现在已经培养超过12个月并定期传代。细胞的上皮性质已通过抗角蛋白抗体染色证实。通过细胞合成低水平刷状缘肽酶和双磷酸酶的能力证明了细胞的肠起源。通过向培养基中加入丁酸钠或佛波醇肉豆蔻酸酯乙酸酯来调节这些酶的表达水平，这导致肽酶的合成增加和双磷酸酶的合成减少。细胞在允许温度（33 ℃）下持续增殖，但在非允许温度（39.5 ℃）下停止增殖。据我们所知，这是第一次从同一小鼠品系的小肠和结肠建立上皮细胞系。这里报道的成功表明，这种转基因小鼠将是一个有用的组织来源的机制，控制肠粘膜细胞的增殖和最终的分化和衰老的研究。这些小鼠也将是一个有用的细胞来源，用于尝试培养来自其他组织的细胞，这些组织已被证明难以在体外培养。
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.