CAJ | 학술논문

目的利用二维差异凝胶电泳(DIGE)技术建立分辨率高和重复性好的人胃癌原发灶与淋巴结转移灶中腺癌细胞的差异蛋白表达图谱,并分析差异表达蛋白质.方法收集、筛选6例新鲜的人胃腺癌原发灶组织及转移的淋巴结组织标本进行比较蛋白质组学研究.用非染色冰冻切片显微切割法分别获取人胃癌原发灶与淋巴结转移灶中的腺癌细胞,裂解后提取蛋白,每例取等量蛋白,任选3例混合,共分成2组.用DIGE染料Cy2、Cy3、Cy5对样品分别标记,然后进行双向电泳,获得人胃癌原发灶与淋巴结转移灶中腺癌细胞的差异蛋白表达图谱,最后使用Typhoon扫描仪进行图像扫描,用DeCyder-Differential分析软件进行分析,识别两者之间的差异表达蛋白质.结果应用非染色冰冻切片显微切割法得到较纯的原发灶与淋巴结转移灶中的癌细胞,不会因为染色问题改变蛋白所带电荷引起的2-DE图像模式的改变,提示它是一种低价、简便的样品制备方法之一.对6例胃癌原发灶与淋巴结转移灶的腺癌细胞样本进行二维差异凝胶电泳,建立了分辨率高、重复性好的差异表达图谱.分析显示差异凝胶1、差异凝胶2的蛋白质点分别为1 416个(相似点1 062个,下调点277个,上调点77个)、1 299个(相似点1 050个,下调点157个,上调点92个).运用DeCyder-Differential软件(Biological Variation Analysis version 5.0)对Gel1、Gel2中的蛋白质点进行分析(P=0.05,阈值:＞1.5倍或者＜-1.5倍),获得11个差异表达的蛋白质.结论非染色冰冻切片显微切割法及DIGE技术是一种简单可行的蛋白质样品纯化方法,建立了分辨率高、重复性好的人胃癌原发灶与淋巴结转移灶中腺癌细胞的差异蛋白表达图谱,为进一步探讨胃癌转移机制、特异性标志物筛选奠定了初步基础.
목적 이용이유차이응효전영(DIGE)기술건립분변솔고화중복성호적인위암원발조여림파결전이조중선암세포적차이단백표체도보,병분석차이표체단백질.방법 수집、사선6례신선적인위선암원발조조직급전이적림파결조직표본진행비교단백질조학연구.용비염색빙동절편현미절할법분별획취인위암원발조여림파결전이조중적선암세포,렬해후제취단백,매례취등량단백,임선3례혼합,공분성2조.용DIGE염료Cy2、Cy3、Cy5대양품분별표기,연후진행쌍향전영,획득인위암원발조여림파결전이조중선암세포적차이단백표체도보,최후사용Typhoon소묘의진행도상소묘,용DeCyder-Differential분석연건진행분석,식별량자지간적차이표체단백질.결과 응용비염색빙동절편현미절할법득도교순적원발조여림파결전이조중적암세포,불회인위염색문제개변단백소대전하인기적2-DE도상모식적개변,제시타시일충저개、간편적양품제비방법지일.대6례위암원발조여림파결전이조적선암세포양본진행이유차이응효전영,건립료분변솔고、중복성호적차이표체도보.분석현시차이응효1、차이응효2적단백질점분별위1 416개(상사점1 062개,하조점277개,상조점77개)、1 299개(상사점1 050개,하조점157개,상조점92개).운용DeCyder-Differential연건(Biological Variation Analysis version 5.0)대Gel1、Gel2중적단백질점진행분석(P=0.05,역치:＞1.5배혹자＜-1.5배),획득11개차이표체적단백질.결론 비염색빙동절편현미절할법급DIGE기술시일충간단가행적단백질양품순화방법,건립료분변솔고、중복성호적인위암원발조여림파결전이조중선암세포적차이단백표체도보,위진일보탐토위암전이궤제、특이성표지물사선전정료초보기출.
Objective To establish differential protein expressing profiles of human gastric adenocarcinoma cell in primary or metastatic lymph node tissues by two-dimensional differential gel electrophorosis (2-D DIGE) so as to investigate the metastatic molecular mechanism of the gastric cancer.Methods After obtaining 6 samples of human primary gastric cancer and metastatic lymph node tissues,with manual no-staining frozen sections microdissection,human gastric adenocarcinoma cells from primary or metastatic lymph node tissues were isolated,and then the total proteins were extracted and purified.Highly sensitive 2-D DIGE was used to separate the total protein differentially expressed in the cells.The proteins were visualized by using a fluorescence scanner at appropriate wavelengths for Cy2,Cy3 and Cy5 dyes (Typhoon 9400).Image analysis was carried out with the DeCyder-Differential analysis software (Biological Variation Analysis version 5.0).Results Not only can the study procure defined adenocarcinoma cell populations from gastric primary or metastatic lymph node tissues,but also can resolve the problem of the change in 2-D DIGE patterns because of the varying in protein changes owing to dyeing.All these showed that the technique was simple,easy to perform,versatile and of particular usefulness when laser capture microdissection (LCM) was practically unavailable.The 2-D DIGE patterns with high resolution and reproducibility from adenocarcinoma cells in gastric primary or metastatic lymph node tissues were obtained.The number of spots in Gel1,Gel2 were 1 416 (similar 1 062,decrease 277,increase 77),1 299 (similar 1 050,decrease 157,increase 92),respectively.A total of 11 differential proteins were acquired by image analysis with DeCyder-Differential analysis software (Biological Variation Analysis version 5.0).Conclusions In this report,a simple,easy to proform method of protein epuration,manual no-staining frozen sections microdissection is described,and have used the highly sensitive 2-D DIGE for the identification of proteins differentially expressing in human gastric adenocarcinoma cells from primary or metastatic lymph node tissues.These results provide a fundamental basis for further study of metastatic mechanism of gastric cancer and screen its specific markers.