中国图书资料分类号R735.2

摘要

目的建立优化的mRNA差示PCR条件；运用建立的条件及GQS-9600分离胃癌GC7901与胃粘膜GES-1细胞株之间的差异表达基因片段；根据获取的序列，设计引物并运用于胃癌组织、血、活检胃粘膜标本检测，拟从中筛选出检出率与符合率高的数对引物，建立胃癌基因诊断方法.

方法以胃癌GC7901与胃粘膜GES-1细胞株为研究对象，探索mRNA差示PCR的最佳反应条件，运用优化的条件分离细胞株之间的差异表达基因片段；以回收的片段作探针，打点杂交证实为真正的差异片段后，对纯化产物进行直接序列分析及GenBank同源性检索，同源性低的序列递交给GenBank；设计引物，采用RT-PCR方法对胃癌组织、血、活检胃粘膜进行检测，筛选出检出率与符合率高的5对引物建立多重PCR诊断胃癌方法.

结果①优化的差示PCR条件为：前5轮PCR循环采用94℃ 35s，40℃ 2min，72℃ 30s，后35轮PCR循环采用94℃ 45s，55℃ 2min，72℃ 1min，最后在72℃延伸7min；②确认并分离出差异条带154条，胃癌细胞株泳道中存在而胃粘膜细胞株泳道中缺失的有82条，胃粘膜细胞株泳道中存在而胃癌细胞株泳道中缺失的有35条，胃癌细胞株泳道中表达高于胃粘膜细胞株泳道中的有23条，胃粘膜细胞株泳道中表达高于胃癌细胞株泳道中的有14条，分别来自于H-T₁₁G，H-T₁₁A，H-T₁₁C锚定引物的差异条带为61条，47条及46条；③从胃癌细胞株泳道中存在而胃粘膜细胞株泳道中缺失的82条带中随机挑选出17个差异片段作探针，进行打点杂交，证实在两细胞株之间呈差异表达；④对17个片段进行直接测序及同源性检索分析，其中7个新序列被GenBank接受，接受号为AF071052至AF071058；⑤GCYS-1，GCYS-4，GCYS-8，GCYS-10，GCYS-11号序列引物在26例胃癌标本中检出覆盖率为100%，在9例病理确诊的胃癌患者活检胃粘膜中检出率为100%，在17例病理确诊为胃癌患者血标本中检出率为53%，在其他组织中检出率较低.

结论①建立了优化的mRNA差示PCR技术的条件；②分离出了154个差异表达的基因片段；③对17个差异片段进行了测序及分析，其中7个新序列被GenBank接受；④建立多重PCR诊断胃癌方法.

Differentially expressed genes were

isolated in gastric carcinoma by

optimized differential display PCR

CUI Da-Xiang, YAN Xiao-Jun and SU Cheng-Zhi

Chinese PLA Institute of Gene Diagnosis of the Fourth Military Medical University, Xi'an 710033, Shaanxi Province, China

Subject headingsstomach neoplasms/diagnosis; RNA, messenger; polymerase chain reaction; gene expression; gene diagnosis

Abstract

AIMTo set up optimized condition of mRNA DD-PCR and to isolate messenger RNA differentially expressed in cell line GC7901 or GES-1; so as to establish gene diagnosis method for gastric cancer.

METHODSBy using cell line GC7901 and GES-1 as research targets, the optimized condition of mRNA DD-PCR was explored. Under optimized condition, cDNA fragments differentially expressed in GC7901 or GES-1 were isolated. Among these fragments 17 cDNA fragments were hybridized to total RNA of cell line GC7901 and GES-1, which were then sequenced and undertaken GenBank similar examination. By using obtained sequences, 17 pairs of primers were designed, and these primers were used to examine clinical samples.

RESULTSOptimized cycle parameters of mRNA DD-PCR for the first 5 round cycles were 94℃35s, 40℃2min; 72℃30s; and for the following 35 round cycles 94℃45s, 55℃2min; 72℃ 1min. By optimized mRNA DD-PCR and GQS-9600 type image and gene quantitive analysis system, 154 bands of cDNA fragments were obtained differentially expressed in cell line GC7901 or GES-1. Seventeen cDNA fragments were hybridized to total RNA of cell lines GC7901 and GES-1, which confirmed that 17 fragments were differentially expressed in GC7901 or GES-1. Sequencing and GenBank searching confirmed that 10 sequences were partially similar to sequences in GenBank, 7sequences were new sequences and accepted by GenBank, these accepted numbers were AF071052 to AF071058. By examining clinical samples, 5 pairs of primers with higher detection rates were screened out.

CONCLUSIONReliable optimized condition of mRNA differential display-PCR is established. 154 bands of fragments differentially expressed in GC7901 or GES-1 were isolated, of which 17 fragments were sequenced and 7 sequences among them were accepted by GenBank; 5 pairs of primers with higher detection rates in clinical samples were screened out and gene diagnosis method of gastric cancer is established.

0引言

mRNA差示PCR(mRNA differential display PCR, DD-PCR)是目前筛选差异表达基因最有效的方法. 自1992年由Liang et al建立以来，已经筛选出一些与肿瘤等疾病相关的基因，显示出广阔的应用前景. 但是此技术存在一些不足之处，假阳性率高，后续处理繁琐等. 我们以胃癌细胞株GC7901与胃粘膜细胞株GES-1为研究对象，优化差示PCR条件，运用建立的条件筛选胃癌差异表达基因片段，利用获取的序列设计引物并运用于临床标本检测，拟建立胃癌基因诊断方法，为临床进一步开展胃癌基因诊断奠定基础.

1材料和方法

1.1材料①细胞株 胃癌 7901细胞株由第四军医大学动物实验中心提供，胃粘膜GES-1细胞株购自北京市肿瘤防治研究所. ②GQS-960型成像及基因分析系统系本所研制，PCR扩增仪系美国Barnstead公司产品. ③PCR产物纯化试剂盒及测序试剂盒分别是Promega公司的wizard^TM PCR preps DNA纯化试剂盒，fmol^TM sequencing kit，丙烯酰胺、双丙烯酰胺、尿素、Tris碱、EDTA皆购自华美公司. ④α-³²P dATP，γ-³²P dATP皆购自北京福瑞公司，PCR试剂系本所提供. ⑤26例胃癌及癌旁组织标本由西京医院王岭教授提供；9例胃癌患者活检胃粘膜标本由西安市中心医院提供；17例胃癌患者血标本由唐都医院张贺龙提供. ⑥差示PCR引物：采用GenHunter公司1996-04提供的序列，Venderbilt大学合成. 5’端引物为8条，3’端引物为3条，它们的序列为：

H-AP₁:5'-AAGCTTGATTGCC-3',

H-AP₂:5'-AAGCTTCGACTGT-3'

H-AP₃:5'-AAGCTTTGCTCAG-3',

H-AP₄:5'-AAGCTTCTCAACG-3'

H-AP₅:5'-AAGCTTAGTAGGC-3',

H-AP₆:5'-AAGCTTGCACCAT-3'

H-AP₇:5'-AAGCTTAACGAGG-3',

H-AP₈:5'-AAGCTTTTACCGC-3'

H-T₁₁G:5'-AAGCTTTTTTTTTTTG-3'

H-T₁₁A:5'-AAGCTTTTTTTTTTTA-3'

H-T₁₁C:5'-AAGCTTTTTTTTTTTC-3'

1.2方法①常规培养胃癌7901细胞株，按文献［1］培养胃粘膜GES-1细胞株. ②采用异硫氰酸胍一步法分离细胞株、组织及血标本中的总RNA，用无RNase的DNase去除其中的DNA，紫外扫描定量后稀释成0.1g/L，于-20℃保存. ③mRNA反转录 以H-T₁₁M(M=G,C,or A)，为起始引物，依次加入无RNase的水9.4μL，5×RT Buffer 4.0μL，dNTP (250μmol/L) 1.6μL，总RNA(0.1g/L)2.0μL，H-T₁₁M 2.0μL，反应条件为：65℃ 5min，37℃， 60min，75℃ 5min，MMLV反转录酶(10⁸U/L)在37℃ 10min时加入. ④PCR扩增 标记好Eppendorf管，依次加入ddH₂O 9.2μL，10×PCR buffer 2μL， dNTP (25μmol/L) 1.6μL，H-AP(1-8) 2.0μL，H-T₁₁M 2.0μL，反转录产物2.0μL，α-³²PdATP 0.5μL，混匀后加入1.5U Taq酶，最后加入30μL石蜡油. ⑤PCR反应条件：前5个循环条件为94℃ 35s，40℃ 2min，72℃ 30s；后35个循环条件为94℃ 45s，55℃ 2min， 72℃ 1min，最后在72℃延伸7min. ⑥60g/L测序胶电泳：配制60g/L聚丙烯酰胺凝胶，充分聚合2h后，预电泳30min，