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[人胃癌下调cDNA片段的克隆及初步表达分析]

[Cloning and primary expression analyses of down-regulated cDNA fragment in human gastric cancer].

作者信息

Wang G, Wang M, You W, Li H

机构信息

Department of Gastroenterology, General Hospital of Chinese PLA, Beijing, 100853 P.R. China.

出版信息

Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2001 Feb;18(1):43-7.

Abstract

OBJECTIVE

To identify human gastric cancer related genes.

METHODS

Specimens of paired tumor, paratumor and normal gastric mucosa tissues were collected from five patients (male 3, female 2, with average age 48.8 +/- 18.1 years) who suffered from stomach antrum adenocarcinoma. Total RNA samples were extracted from these specimens, then studied by fluorescent differential display reverse transcription polymerase chain reaction (DDRT- PCR) analysis. The differentially expressed bands of interest were recovered, purified and cloned, then they were analyzed by sequencing, Northern blot and RT-PCR. Through BLAST, the sequencing results were compared with GenBank database for homology analysis.

RESULTS

One of the interesting cDNA bands expressed much lower in all five tested tumor samples than in their normal and paratumor counterparts. This band was named W4. Northern blot analysis showed a consistent result with that of DDRT-PCR. W4 was cloned into pGEM-T easy vector. Sequence analysis showed that W4 consists of 712bp, this sequence was named W44. BLAST analysis revealed that W44 has extremely low sequence identity with any genes from GenBank and any sequences from EST database. This sequence data was submitted to GenBank with accession No. AF150631. RT-PCR analysis showed that W4 was expressed much lower in 11/15 gastric cancer tissue than in paratumor and normal samples.

CONCLUSION

A novel cDNA sequence related to human stomach adenocarcinoma was identified.

摘要

目的

鉴定与人类胃癌相关的基因。

方法

从5例胃窦腺癌患者(男3例,女2例,平均年龄48.8±18.1岁)中收集配对的肿瘤、癌旁和正常胃黏膜组织标本。从这些标本中提取总RNA样本,然后通过荧光差异显示逆转录聚合酶链反应(DDRT-PCR)分析进行研究。回收、纯化并克隆感兴趣的差异表达条带,然后通过测序、Northern印迹和RT-PCR进行分析。通过BLAST将测序结果与GenBank数据库进行比较以进行同源性分析。

结果

在所有5个测试的肿瘤样本中,有一个感兴趣的cDNA条带的表达明显低于其正常和癌旁对应组织。该条带被命名为W4。Northern印迹分析结果与DDRT-PCR一致。W4被克隆到pGEM-T easy载体中。序列分析表明W4由712bp组成,该序列被命名为W44。BLAST分析显示W44与GenBank中的任何基因以及EST数据库中的任何序列的序列同一性极低。该序列数据已提交至GenBank,登录号为AF150631。RT-PCR分析表明,W4在11/15的胃癌组织中的表达明显低于癌旁和正常样本。

结论

鉴定出一个与人类胃腺癌相关的新cDNA序列。

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