肝细胞癌中一种高通量二维细胞识别技术方法的建立  

A high-throughput two-dimensional screening technique for cellular recognition and localization in hepatocellular carcinoma

作　　者：朱玉珍[1] 符达 刘黎黎[2] 马雨水[2] 沈锡中[2] 陈锡美[1] 

机构地区：[1]上海市同济大学附属同济医院消化内科,上海市200065 [2]复旦大学附属中山医院消化科,上海市200032

出　　处：《世界华人消化杂志》2011年第23期2455-2460,共6页World Chinese Journal of Digestology

基　　金：中国博士后基金面上项目;No.20100480542~~

摘　　要：目的:建立一种新颖的高通量二维细胞识别、定位技术,为肝癌干细胞的鉴定、异质性分化细胞分类等提供技术平台.方法:收集原发性肝细胞癌患者手术标本1例,每个标本分别石蜡包埋,以1μm的厚度连续切5片.每张切片取第一片做常规HE染色,病理诊断.其余4张切片分别以肝癌组织中目前已报道的8个可能的肝癌干细胞标志物进行了免疫荧光标记,利用试剂Hoechst 33342(蓝色)标记细胞核定位,异硫氰酸荧光素(fluoresce iniso thio cyanate,FITC),四甲基异硫氰酸罗丹明(tetramethy l rhodamine isothiocyanate rhodamine)双荧光标记待测抗体,荧光显微镜检测荧光位置及强度.结果:以4个切片中均能识别的细胞核做定位,确定每个切片1个1×100显微镜视野中有效细胞的个数,作出4个切片合并的细胞图谱,建立了一种新颖的二维细胞识别技术.检测得到有效细胞为2772个.检测的8个抗体均在肝癌细胞中表达,并且不同肝癌细胞中肝癌干细胞标志物的表达情况不同,同一肝癌细胞可同时表达多个肝癌干细胞的分子标志,且可见8个抗体均阳性的肝癌细胞.其中2772个有效细胞中有2453个细胞为阴性,所占比例为884.9‰.结论:完成了高通量二维细胞识别、定位技术方法的建立,该方法可在一个肝癌干细胞上检测两种以上的肝癌干细胞分子标志的表达情况.AIM： To establish a novel high-throughput two- dimensional screening technique for cellular recognition and localization in hepatocellular carcinoma （HCC）. METHODS： HCC specimen was collected from a patient who underwent radical resection. The paraffin-embedded specimen was serially sectioned at a thickness of 1 μm. Five serial sections were used for staining： one for H＆E staining and the other four for immunofluorescence staining for detecting eight reported liver cancer stem cell （LCSC） markers. Fluorescein isothiocyanate （FITC） and tetramethyl rhodamine isothiocyanate （rhodamine） were used for fluorescent imaging for double staining. The sections were counterstained with Hoechst33342 to demonstrate the nuclei for cellular localization. Fluorescence microscopy was used to detect the fluorescence intensity and localization. RESULTS： We identified the valid number of cells in a visual field （1 × 100） of microscopy and delineated the merged cell map. The results showed that 8 LCSCs biomarkers could be detected in 2 772 valid cells. The expression levels of biomarkers were different in these cells and a single valid cell could express 0-8 biomarkers. No biomarkers could be detected in 2 453 cells （88.5%）. CONCLUSION： A high-throughput two-dimensional screening technique for cellular recognition and localization has been successfully developed and can be used to detect the expression of two or more LCSC markers in one liver cancer cell.

关 键 词：肝癌干细胞 高通量 二维技术 细胞识别 细胞定位 

分 类 号：R735.7[医药卫生—肿瘤]