摘要
目的:食管癌是严重威胁人类健康及生命的恶性肿瘤之一。我国是食管癌高发区,食管癌的发病率居世界第一位。目前食管癌的早期诊断水平仍欠理想,大部分患者发现时已到中晚期。当今治疗食管癌的方法主要是外科手术结合放、化疗的综合治疗,但食管癌治疗效果仍不理想,5年生存率低于30%。如何早期发现食管癌,及尽早控制食管癌癌前病变的发展,提高生存率,延长食管癌患者生存时间,一直是医疗工作者关注的课题之一。再生因子Iα(Regeneration gene Iα, Reg Iα)属于一种钙依赖性植物血凝素家族( c型植物血凝素家族),它是一组分泌蛋白,其功能相当于急性反应蛋白,抗凋亡因子或胰腺β细胞和神经细胞分泌的生长因子,多种功能与肿瘤的发生、发展、转移关系密切。本实验应用定量聚合酶链反应、免疫组织化学和ELSIA的方法探讨RegIα在正常食管黏膜和食管鳞癌组织中的表达差异,与临床病理学特征的关系及手术前后患者体内Reg Iα的表达水平的变化。意义在于研究RegIα在食管癌病人中的表达情况,探讨其表达的临床意义。
方法:⑴食管癌组织标本共30例,来自河北省人民医院胸外科、河北医科大学第四医院胸外科,2008年10月-12月的食管癌手术标本(病理证实均为鳞状细胞癌),病理诊断为正常的食管黏膜20例作为正常组织对照,取自食管癌组织手术远端5cm。应用半定量聚合酶链反应(Reverse transcriptase polymerase chain reaction ,RT-PCR)、免疫组织化学和ELSIA的方法检测Reg Iα的表达。
应用RT-PCR的方法检测RegIαmRNA的表达,首先提取食管癌和食管正常黏膜组织的总RNA,普通琼脂糖凝胶电泳测定RNA的完整性。按RT-PCR试剂盒说明制备cDNA,配置20μlPCR反应体系,加入自100ng cDNA样本及合成引物进行扩增,应用凝胶成像仪上扫描记录各特异性DNA扩增片段灰度,比较目的基因与内参基因GAPDH的扩增片段灰度,用其比值作为mRNA表达水平的相对参数。
应用免疫组织化学的方法检测RegIα蛋白在食管癌组织中的表达;根据0 ----3+的评分标准:0---不显色或少于10%的肿瘤细胞浆呈现任何水平的阳性染色;1+----超过10%的肿瘤细胞浆呈现微弱染色,或在部分细胞浆上染色;2+----超过10%的肿瘤细胞浆全部弱染色;3+----超过10%的肿瘤细胞浆全部呈现强染色。
⑵患者血液标本来源:30例食管癌手术病人术前1天和术后7天采新鲜血,经离心获得血清;20个健康员献血员血清作为正常对照。
应用ELSIA方法分析食管癌患者血液中RegIα的表达,结果判断:每个标准品和标本的OD值应减去零孔的OD值,按标准品浓度及吸光度做出标准曲线,根据各待测样品吸光度在标准曲线上的位置读出相应浓度。统计学方法:应用SPSS 13.0统计学软件对所有数据进行t检验、χ2检验或秩和检验分析,计算P值,以P值<0.05判断差异有统计学意义。
结果:
1 RegIα蛋白在正常食管黏膜及食管癌组织中的表达情况:在20例正常食管黏膜组织中,RegIα表达均为阴性,但在30例食管癌组织中表达阳性率为60%(18/30)。统计学处理显示,两组表达的阳性率差异明显(P<0.05)。
2 RegIαmRNA在正常食管黏膜与食管癌组织中表达情况:食管癌中的Reg IαmRNA的表达强度明显高于正常食管黏膜。两组间Reg IαmRNA的表达水平存在明显的差异,RegIαmRNA表达的相对灰度值分别为:1.132±0.247(食管癌组织),0.719±0.176(正常食管粘膜);统计学处理显示:差异性显著(P<0.001)。
3 RegIα的表达水平与食管癌病理学特征的关系:根据患者术后的病理学特征进行分组,患者RegIα水平与肿瘤的大小、侵润深度无明显相关(P>0.05);但与分化程度与肿瘤是否转移明显相关(P<0.001)。
4 RegIα在食管癌患者血清中的表达水平明显高于正常对照组,P=0.011;且其表达水平在食管癌切除后明显下降,P=0.03。
结论:
1 RegIα蛋白在正常食管黏膜无表达,在食管癌中高表达。
2 RegIα与食管癌的分化、转移明显相关,其表达可能介入食管癌细胞的分化及转移机制之中。未发现RegIα与食管癌其它病理特征相关。
3食管癌病人RegIαmRNA的表达水平显著高于食管正常黏膜,故可作为食管癌早期检测的标志物。
4 RegIα在患者食管癌切除前后表达水平明显变化,所以它的表达与食管癌发生有关。
Objective: Esophageal cancer is one of the cancers that seriously threat the human health and life, the incidence of esophageal cancer is high in my country ,the morbidity ranks first in the world, and the mortality rates is second only to that of gastric cancer and lung cancer . The level of the early diagnosis of esophageal cancer is still unsatisfactory at present, most patients had been the middle and late stage when the disease had been found. The current method of treatment of esophageal cancer is mainly surgery combined with radiotherapy and chemotherapy , but the survival rate is still unsatisfactory and 5-year survival rate of less than 30%. How to early detect esophageal cancer and precancerous lesions and how to early control the development of esophageal cancer, early how to improve survival rates and prolong survival time of patients has always been a matter that health care workers concern.Regenerating factor Iα(Reg Iα) is one of the calcium-dependent lectin family which includes a group of secreted proteins. They are secreted by pancreatic andβcells nerve cells and the digestive system epithelial cells at acute phase response, with a variety of functions with the tumor occurrence, development, transfer. In this study, the expression of the Reg Iαin normal esophageal mucosa and esophageal squamous cell carcinoma was study by quantitative polymerase chain reaction(PCR) and immunohistochemistry method and the method of ELSIA and analys the relationship between the differences expression of the Reg Iαand the clinical pathological features,Before and after surgery in patients with in vivo Reg Iαexpression levels change. And explore the clinical significance of its expression
Methods : We collected 30 specimens of the esophageal cancer from the Hospital of Hebei Province People and Hebei Medical University 4th Hospital at the time Oct—Dec in 2008. The specimens confirmed by pathology were squamous cell carcinoma, 20 cases of normal esophageal mucosa as normal tissue controls. Semi-quantitative polymerase chain reaction ( RT-PCR) and immunohistochemistry was used to detect the expression of Reg Iα. The expression of Reg IαmRNA was detected by RT-PCR , after the esophageal cancer and esophageal normal mucosa of total RNA extracted, the RNA integrity was determinated by agarose gel electrophoresis. Prepared cDNA according to RT-PCR kit instructings , configured 20μl PCR reaction system adding self-100ng cDNA samples、adding primers amplified、gel-imaging device to scan、recorded the specific DNA fragment amplified gray、compared the fragment grayscale of target gene and the GAPDH gene amplification, its ratio as the relative expression levels of mRNA . The expression of Reg Iαin esophageal tissues was detected by Immunohistochemical method ; according to 0 ---- 3 + rating criteria :0 --- no color or less than 10% of the tumor cells showed positive staining ; 1 +---- more than 10% of the tumor cells showed weak staining partly ; 2 +---- more than 10% of the tumor cells showed the weak staining; 3 +---- more than 10% of the All tumor cells showed strong staining.
The source patient's blood samples: 30 patients blood with esophageal cancer were abtained before surgery a day and seven days after operation, obtained serum after centrifugation;20 healthy blood donors serums as normal controls.
ELISA was used to examine the expression level of regenerating fator Iα(Reg Iα)in the patients with esophageal cancer .Evaluation of results: Subtract the average zero standard optical density to the readings for each standard, control, and sample. Create a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The samples read from the standard curve acording the optical density .
Statistical Methods: all data were analysed by SPSS13.0 statistical software forχ2 test or rank sum tests and t tests, P value <0.05 was statistically significant.
Results:
1 The expression of Reg Iαin normal esophageal mucosa and esophageal cancer tissues : the expression of Reg Iαwere negative in 20 cases of normal esophageal mucosa, but in 30 cases of esophageal cancer tissues the positive rate was 60% (18/30 ). The positive rate of Reg Iαwas significantly differences between the two groups (P <0.05).
2 The expression of Reg IαmRNA in the normal esophageal mucosa and esophageal cancer tissues:The expression of Reg IαmRNA in esophageal was significantly higher than the normal esophageal mucosa. the two groups Reg IαmRNA expression gray scale values were: 1.132±0.247 (esophageal cancer), 0.719±0.176 (normal mucosa); there is significant differences at the expression level ,(P <0.001).
3 The relationship between the expression level of Reg Iαand the pathological features of esophageal cancer: Divided the patients into different groups according to the pathological features, there were no significant correlation between the expression level of Reg Iαand tumor size, invasion depth of tumor (P> 0.05); but there were significantly correlated with the degree of differentiation and the tumor metastasis (P < 0.001).
4 The expression level of RegIαin patients’serum with cancer of the esophagus obviously higher than the normal control group, P=0.011; And the expressesion level were to obviously dropped after cancer of the esophagus excised, P=0.03.
Conclusions:
1 The expression of Reg Iαwas highly expressed in esophageal cancer and negative expression in normal esophageal mucosa.
2 The expression of Reg Iαwas significantly correlated with esophageal cancer differentiation and metastasis , its expression may be involved in esophageal cancer cell differentiation and transfer mechanism. No found the association between the expression of Reg Iαand the other pathological features of esophageal cancer.
3 Reg IαmRNA expression in esophageal cancer were significantly higher than that in normal esophageal mucosa, it can be used as a marker for early detection of esophageal cancer.
4 The expression level of RegIαin patients’serum was significantly changed after resected the esophageal cancer ,so its expression was related with the occurrence of esophageal cancer.
引文
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