Analysis of genomic changes in the intestinal metaplastic epithelial cells of the human stomach

1)本项目最重要的成果之一是，在组织RNA保存良好的条件下，我们开发了一种非常有效的方法来分离化胃腺的最小靶点。用隐窝分离技术分离幽门粘膜中的腺体，在70%的乙醇中固定20-30分钟，然后用阿尔辛蓝(AB)染色鉴定杯状细胞。除了在相差显微镜下定义离体腺的形态特征外，AB染色还使我们能够区分胃型和肠型腺体。用RT-PCR方法成功地从已完成的分离腺体中合成了CDNA。2)然而，由于仅从一个分离腺体合成足够的CDNA太困难，不能实现差异显示。3)每个肠化生(IM)亚型的分离腺体收集10个并进行基因组分析。具有三个子类型的…用半定量逆转录聚合酶链式反应(RT-PCR法)检测更多的离体腺(IM(-)、中度IM和重度IM)中同源盒基因CDX-1和CDX-2的mRNAs在隐窝未成熟细胞中诱导和维持大肠粘膜上皮细胞分化的重要转录因子的表达，并以β-肌动蛋白mRNA的表达作为阳性对照。肠化的胃腺持续表达CDX-1和CDX-2mRNAs，其表达水平一般随IM的严重程度而增加。CDX-1和CDX-2在mRNA水平上无明显差异。此外，当检测MUC5AQ Pg11、MUC2、Villin、Defensins的mRNA水平时，未合并肠化、伴胃肠混合型肠化和肠型肠化的腺体表现出不同的表达模式。对其基因产物的免疫组织化学分析表明，它们之间的表达密切相关。4)综上所述，一些在肠粘膜上皮细胞分化和维持中起重要作用的基因可能控制着胃粘膜中IM的出现。5)这些基因之间的相互关系正在研究中。较少

1) Qie of the most important results of this project is that we couldexploit a very effective method to isolate the smallest target of mtestinalized gastric glands under conditions in which the tissue RNA was well preserved After glands in pyloric mucosa were isolated by a crypt isolation technique, they were fixed in 70 % ethanol in 20-30minutes and then stained by Alcian-blue (AB) staining to identify goblet cells. In addition to definition of morphological characteristics of the isolated glands under phase-contrast microscopy, AB staining allowed us to discriminate between gastric and intestinal type glands. Synthesis of CDNA was successfully from accomplished isolated glands using the RT-PCR method.2) However, differential display could not be achieved because it was too difficult to synthesize sufficient CDNA from only one isolated gland.3) Isolated glands of each subtype of intestinal metaplasia (IM), ten were collected and processed for genomic analyses. With the three subtypes … More of isolated glands (IM (-), moderate IM, and severe IM), the expression of mRNAs of homeobox genes, Cdx-1 and Cdx-2, revealedto be important transcriptional factors introducing and maintaining the differentiation of large or small intestinal mucosal epithelial cells in the immature cells of the crypts, were investigated by the semi-quantitative RT-PCR method The expression of β-actin mRNA was also examined as a positive control. The intestinalized gastric glands consistently expressed both Cdx-1 and Cdx-2 mRNAs, levels generally increasing with severity of IM. No obvious differences in mRNA levels between Cdx-1 and Cdx-2 were apparent. Moreover, when mRNA levels of MUC5AQ Pgll, MUC2, villin, defensinS were examined, isolated glands without IM, associated with gastric and intestinal mixed-type IM, and intestinal-type IM showed different expression patterns. Immunohistochemical analyses with antibodies to their gene products demonstrated close correlation between mRNA and protein expression.4) In conclusion, it is suggested that some genes, which have important roles in differentiation and maintenance of the intestinal mucosal epithelial cells, might control the appearance of IM in gastric mucosa.5) The interrelationships among these genes are now under investigation. Less