摘要目的筛选数个在胃癌及癌前病变粘膜中检出率高的差异表达基因片段并探讨其临床意义.方法以筛选出的gcys-1至gcys-18的新基因片段作为研究对象，采用定量RT-PCR技术检测48例胃癌、16例其他肿瘤及110例活检的胃粘膜标本.结果①在48例胃癌中，表达检出率高的6个片段依次为：gcys-10为90.5%，gcys-1为80.9%，gcys-18为69.5%，gcys-11为61.9%，gcys-4为42.8%，gcys-8为35.7%，在癌旁组织中检出率＜10%；6个片段的检出率在胃癌及癌旁组织中存在显著性差异(P＜0.01)；②在16例其他肿瘤中，表达率依次为：gcys-10为37.5%，gcys-1为14.3%，gcys-18为27.5%，gcys-11为43.7%，gcys-4为14.3%，gcys-8为31.3%；③33例萎缩性胃炎中，表达率依次为：gcys-10是40.5%，gcys-1是16.7%，gcys-18为44.5%，gcys-11为43.3%，gcys-4为33.3%，gcys-8为48.3%；28例肠腺化生标本中，表达率依次为：gcys-10是60.5%，gcys-1是35.3%，gcys-18为34.5%，gcys-11为58.8%，gcys-4为23.8%，gcys-8为58.3%；17例萎缩性胃炎合并肠腺化生标本中，gcys-10是70.5%，gcys-1是36.7%，gcys-18为54.5%，gcys-11为63.3%，gcys-4为37.8%，gcys-8为68.3%；在19例不典型增生标本中，表达率依次为：gcys-10是50.8%，gcys-1是39.3%，gcys-18为34.5%，gcys-11为48.8%，gcys-4为25.8%，gcys-8为48.4%；在6例胃溃疡、7例急性胃炎中，gcys-10为7.5%，gcys-1为8.0%，gcys-18为7.5%，gcys-11，gcys-4，gcys-8表达率皆为0；这6个基因片段检出率在癌前病变与胃溃疡、急性胃炎中存在显著性差异(P＜0.01).结论gcys-1，4，8，10，11，18这6个基因片段可能与胃粘膜癌前病变、早期胃癌发生发展有关，监测癌前病变粘膜中此6个基因的表达水平的变化可能对胃癌的早期发现更有意义；此研究为建立胃癌预警系统打下基础.

中国图书馆分类号R 735.2

Screening and its clinical significance of 6 fragments of highly expressing genes in gastric cancer and precancerous mucosa

CUI Da-Xiang¹, YAN Xiao-Jun¹, ZHANG Li², ZHAO Jing-Rong¹, JIANG Mei², GUO Yan-Hai¹, ZHANG Ling-Xia², BAI Xi-Ping² and SU Cheng-Zhi¹

¹Chinese PLA Institute of Gene Diagnosis, Fourth Military Medical University, Xi'an 710033, Shaanxi Province, China

²Department of Gastroenterology, Xi'an Municipal Central Hospital, Xi'an 710003, Shaanxi Province, China

AbstractAIMTo screen several genes which were differentially expressed in gastric cancer and precancerous pathological changes with higher examination rates.METHODSBy using gcys-1 to gcys-18 as research targets, 48 specimens of gastric cancers and 16 specimens of other tumors as well as 110 specimens of biospied gastric mucosa were screened by quantitative RT-PCR.RESULTSIn 48 specimens of gastric cancers, 6 fragments with higher examination rates were gcys-10 (90.5%), gcys-1 (80.9%), gcys-18 (69.5%), gcys-11 (61.9%), gcys-4 (42.85%) and gcys-8 (35.7%). Their positive rates in paratumor tissues were ＜10%, with a significant difference between gastric cancers and paratumor tissues (P＜0.01). In 16 specimens of other tumors, the expression rates of gcys-10, 37.5%; gcys-1, 14.3%;gcys-18, 27.5%; gcys-11, 43.8%; gcys-4, 14.3%; and gcys-8, 31.2%. In 33 specimens of atrophic gastritis, their expression rates were 40.5%, 16.7%, 44.5%, 43.3%, 33.3%, and 48.3%. In 28 specimens of intestinal metaplasia, their expression rates were 60.5%, 35.3%, 34.5%, 58.8%, 23.8%, and 58.3%. In 17 specimens of atrophic gastritis with intestinal metaplasia, the expression rates were 70.5%, 36.7%, 54.5%, 63.3%, 37.8%, and 68.3%. In 19 specimens of dysplasia, the expression rates were 50.8%, 39.3%, 34.5%, 48.8%, 25.8%, and 48.4%. In 6 gastric ulcers and 7 acute gastritis, the expression rates were 7.5%, 8.0%, 7.5%, 0% and 0%. The expression rates of 6 fragments between precancerous changes and gastric ulcer and gastritis were significantly different (P＜0.01).CONCLUSIONgcys-1, 4, 8, 10, 11, 18 may be correlated with precancerous changes and early gastric cancer. To monitor these 6 genes in gastric mucous may be very important to finding early gastric cancer.

Subject headingsstomach neoplasms/pathology; gastric mucous; gene expression; polymera chain reaction; precancerous condition

0引言

胃癌在世界恶性肿瘤中占第2位，在我国居第1位. 胃癌的发生与年龄、性别、社会经济状况、卫生水平及人口居住密度、烟酒嗜好、环境危险因素等有关. 尽管胃癌病因复杂，但是这些因素最终都在不同阶段作用于不同基因，引起相关基因表达水平改变，这些基因共同作用，最终导致胃癌的发生发展. 胃癌差异表达基因gcys-1至gcys-18是我们用DDRT-PCR技术从细胞株GC7901与GES-1中筛选出的胃癌差异表达基因片段. 我们通过对收集的110例活检胃粘膜及48例胃癌等组织进行定量RT-PCR检测，拟筛选出在胃癌及癌前病变组织中检出率高的差异表达基因片段，为建立胃癌的早期预警系统奠定基础.

1材料和方法

1.1材料①胃癌手术标本32例，活检胃癌标本16例；活检胃粘膜标本110例，其中萎缩性胃炎33例，肠腺化生28例，萎缩性胃炎合并肠腺化生17例，不典型增生19例，胃溃疡6例，急性胃炎7例. 绝大部分标本皆收集于西安市中心医院，部分胃癌标本收集于西京医院，5例乳癌、3例肾癌、4例肝癌、1例肺癌及3例食管癌标本由西医二附院陈伟博士提供；所有标本皆经病理HE染色确诊. ②引物由崔大祥博士设计，上海中科院生化所合成，MMLV反转录酶购自Promega公司，PE5700型PCR仪由美国PE公司提供，其他试剂由基因诊断研究所提供. ③胃粘膜GES-1细胞株购自北京市肿瘤防治研究所.

1.2方法①所有标本获取后，立即投入液氮中保存. ②采用Promega公司的PolyATtract^ System 1000试剂盒快速提取组织标本中的mRNA，紫外扫描定量后稀释成0.1 g/ L，立即进行mRNA反转录：以oligo(dT)18为起始引物，依次加入无RNase的水10.4 μL，5×RT Buffer 4.0 μL，dNTP(250 μmol/ L)1.6 μL，mRNA(0.1 g/ L)1.0 μL, oligo (dT)18引物 2.0 μL，反应条件为：65℃ 5min，37℃ 60min，75℃ 5min，MMLV反转录酶(200 MU/ L)在37℃ 10min时加入. ③PCR扩增：取反转录产物2 μL作为模板，依次加入：10×PCR Buffer 3 μL，2.5 mmol/ L dNTPs 3 μL，上游引物各1 μL(10pmol)，下游引物各1 μL(10pmol), ddH₂O 10 μL，混合后加入2.5 U Taq酶. PCR循环条件为：94℃ 35s，55℃ 1min，72℃ 35s，35个循环，最后在72℃延伸7min. ④PCR结果判断标准：在同一条件下，每次PCR以β-actin作内对照，以克隆质粒作阳性对照. PCR产物进行10 g/ L琼脂糖电泳20min后，在GQS-9600型基因定量分析系统中扫描摄入电泳图象后，对特征性条带进行光密度扫描，产物吸光度/对照产物吸光度比值进行成对资料统计学处理.

2结果

2.1差异表达基因片段在肿瘤标本中表达①在48例胃癌中，表达检出率高的6个片段依次为：gcys-10高表达率为90.5%，gcys-1高表达率为80.9%，gcys-18高表达率为69.5%，gcys-11高表达率为61.9%，gcys-4高表达率为42.8%，gcys-8高表达率为35.7%；在癌旁组织中检出率＜10%；这6个基因片段在胃癌组织中表达检出率与癌旁组织中检出率存在显著性差异(P＜0.01). 因此，我们认为此6个基因片段是胃癌高表达基因片段，与胃癌的发生发展可能有关(图1). ②在16例其他肿瘤中，表达率依次为：gcys-10表达率为37.5%，gcys-1表达率为14.3%，gcys-18表达率为27.5%、gcys-11表达率为43.7%，gcys-4表达率为14.3%，gcys-8表达率为31.3%. 因此，我们认为此6个基因片段可能与其他肿瘤有关.

图16个差异表达基因片段扩增产物电泳结果.

①阳性对照；②阴性对照；③PCR Marker;

4～9差异表达基因片段扩增产物，上一排β-actin内对照.

2.2差异表达基因片段在活检非癌胃粘膜中表达在33例萎缩性胃炎中，表达率依次为：gcys-10是40.5%，gcys-1是16.7%，gcys-18表达率为44.5%, gcys-11表达率为43.3%，gcys-4表达率为33.3%，gcys-8表达率为48.3%. 28例肠腺化生标本中，表达率依次为：gcys-10是60.5%，gcys-1是35.2%，gcys-18表达率为34.5%，gcys-11表达率为58.8%，gcys-4表达率为23.8%，gcys-8表达率为58.3%. 在17例萎缩性胃炎合并肠腺化生标本中，gcys-10是70.5%，gcys-1是36.