导向激光捕获显微切割技术在贲门腺癌差异蛋白质组学的应用研究  

Application of navigated laser capture microdissection to differential proteomic analysis of human gastric cardia adenocarcinoma

作　　者：程妍[1] 张军[1] 张蓉[1] 刘冬[1] 陈晓黎[2] 谢丹红[1] 龚均[1] 

机构地区：[1]西安交通大学医学院环境和疾病相关基因教育部重点实验室,陕西西安710061 [2]西安交通大学医学院第二附属医院病理科,陕西西安710004

出　　处：《西安交通大学学报（医学版）》2014年第4期557-561,564,共6页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.30470785)~~

摘　　要：目的建立一套适用于贲门腺癌差异蛋白质组学研究的导向激光捕获显微切割技术(navigated laser capture microdissection,NLCM)方法,并探讨其可行性。方法比较染色剂和固定剂对蛋白质影响的差异,探讨不同处理条件下贲门腺癌和癌旁组织切片的镜下组织学特点,改进常规LCM,建立在染色切片指引下分离细胞的NLCM技术,并通过双向凝胶电泳分析对该技术进行评价。结果染色剂对蛋白质的影响大于700mL/L乙醇固定剂;光镜下在不染色的组织切片上,贲门腺癌细胞、肠化生细胞和正常柱状上皮细胞都有其各自的形态特异性,易于辨认;NLCM可以准确纯化贲门腺癌细胞、肠化生细胞和正常柱状细胞;LCM的苏木素染色贲门腺癌细胞(A组)、NLCM的无染色贲门腺癌细胞(B组)和未切割的新鲜贲门腺癌组织(C组)的双向电泳表达图谱非常相似。各组蛋白质点的数量之间差异无统计学意义(P>0.05)。A组碱性端蛋白等点聚焦较差,部分蛋白质点的表达量减低或消失,B组双向电泳表达图谱蛋白质点分离较好。结论 NLCM不但可以快速、准确地将贲门腺癌及其癌前病变细胞进行分离,而且有效避免了染色剂对蛋白质提取质量的影响,是一项适用于贲门腺癌蛋白质组学研究的样品制备方法。Objective To set up an appropriate method to apply navigated laser capture microdissection （NLCM） to differential proteomic analysis in human gastric cardia adenocarcinoma （GCA） and explore its feasibility.Methods We compared effects of dyeing agents and ethanol fixative agents on the quality of protein extraction and then observed the microscopic histological features of GCA and its adjacent tissue sections.Based on them,we improved the conventional LCM method and established the NLCM,which captured cells guided by the staining slides.Two-dimensional gel electrophoresis （2-DE） was used to evaluate this technique.Results ① Compared with 70％ ethanol fixing agent,dyeing agents showed much greater effects on the quality of protein extraction; ② Malignant,intestinal metaplasia and normal gastric cardiac epithelium cells had their own microscopic histological features,which made them recognized easily on the unstained slides; ③ Malignant,intestinal metaplasia and normal gastric cardiac epithelium cells could be separated accurately by NLCM; ④ the 2-DE patterns of hematoxylin-stained malignant cardiac epithelium cells by LCM （group A）,unstained malignant cardiac epithelium cells by NLCM （group B） and unstained and undissected GCA tissues （group C） were very similar.No significant difference in protein spot number was observed in the three treatment groups （P＞0.05）.Group A showed poor protein focusing at the basic end of the gel,and some of the protein spots decreased and even disappeared.Group B showed a high resolution 2-DE pattern.Conclusion NLCM can not only separate cells rapidly and precisely but also effectively avoid the influences of dyeing agents on the quality of protein extraction.Therefore,it is suitable and feasible for sample preparation in GCA proteomics.

关 键 词：贲门腺癌 激光捕获显微切割 蛋白质组 双向凝胶电泳 

分 类 号：R5[医药卫生—内科学]