Development and Genetic Changes of Isolated Aberrant Crypts Based on Clonal Analysis

1.正常大鼠结肠隐窝的发育及DMH诱导的ACF：研究表明，生后正常大鼠的结肠隐窝是通过分裂机制繁殖的，DMH诱导的ACF也是如此。PhlP诱导的大鼠ACF的分布：胃内注射PhlP诱导的大鼠ACF在治疗后12周和25周主要分布在远端结肠，在治疗后50周主要分布在近端结肠。ACF在远端结肠中的数量随着时间的延长而减少，在近端结肠中的数量随着时间的推移而增加。在50周时，肿瘤主要集中在近端结肠。提示早期远端ACF是可逆的，部分晚期ACF可能已发展为癌变。嵌合小鼠结肠隐窝分离及C3H特异性单抗克隆分析：正常嵌合小鼠单个结肠隐窝呈克隆性，粘膜呈由同一克隆来源的两组隐窝拼接而成的斑片状。就我们的研究而言，所有的隐窝都是由DMH诱导的嵌合小鼠ACF中的一株菌株组成的。嵌合小鼠结肠隐窝的微卫星多态克隆分析：通过聚合酶链式反应，发现一个品系间具有多态性的DNA微卫星标记D8Mit4可以区分嵌合小鼠分离的结肠隐窝的克隆来源。检测结果与CsA染色结果一致。结论：根据上述结果，我们推测ACF是通过分裂机制从单个结肠隐窝单克隆性发育而来的。CsA染色结果证明了这一假设是正确的。从ACF的分布、形态、遗传变化和克隆性等方面进行研究，可以揭示ACF作为结肠癌癌前病变的可逆性和不可逆性。

1. Development of normal colonic crypts and DMH-induced ACF in rats : it was shown that normal postnatal rat colonic crypts reproduced themselves by fission mechanism and as was also the case with DMH-induced ACF.2. Distribution of PhlP-induced rat ACF : Intragastrical administration of PhlP induced rat ACF mainly in the distal colon at weeks 12 and 25 and predominanly in the proximal colon at week 50 after the treatment. The number of ACF decreased in the distal colon and increased in the proximal colon with time. Cancers were mostly localized in the proximal colon at week 50. It suggested that the early distal ACF were reversible and some of late distal ACF might have developed into cancers.3. Colonic crypts isolation and clonal analysis with C3H-specific monoclonal antibody (CSA) in chimeric mice : Normal single colonic crypt of chimeric mice were revealed as clonal and the mucosa showed like a patchwork consisting of two groups of crypts derived from the same clonal origin. As far as we investigated, all crypts were constituted with eighter orgin of a strain in a DMH-induced ACF in chimeric mice.4. Clonal analysis of colonic crypts in chimeric mice by microsatellite polymorphism : Using PCR reaction, a DNA microsatellite marker D8Mit4, polymorphic among strains, could distinguish the clonal origin of isolated crypts of chimeric mice. The result was identical with that judged by CSA staining.5. Conclusion : With the above results, we hypothesized that ACF were monoclonally developed from a single colonic crypt by fission mechanism. The results of CSA staining proved this hypothesis to be true. Investigation of ACF in terms of distribution, morphology, genetic change, and clonality may reveal the character of reverlibility and irreversiblity of ACF as a precancerous lesion of colonic tumor.