LCM联合快速免疫组化技术——一种获取肿瘤原位血管内皮细胞的可行途径

COMBINATION OF QUICK STAINING IMMUNOHISTOCHEMISTRY AND LASER CAPTURE MICRO-DISSECTION—A FEASIBLE APPROACH TO OBTAINING HIGHLY PURE TUMOR VASCULAR ENDOTHLIAL CELLS FROM IN SITU TUMOR TISSUES

目的肿瘤血管内皮细胞对肿瘤发生发展极为重要,是目前肿瘤研究的热点。本研究为从肿瘤组织原位获取高纯度血管内皮细胞进行基因表达研究摸索可行方法。方法获取淋巴瘤组织标本后,置于锌固定液中固定,并通过进行激光捕获显微切割(lasercapture microdissection,LCM)前操作模拟LCM环境,确定锌固定法对RNA完整性的保护作用。将组织标本制作冰冻切片,采用快速免疫组化染色方法标记血管内皮细胞,利用LCM技术获取肿瘤组织原位血管内皮细胞,并用RT-PCR方法对所获细胞进行纯度检测。结果无论是固定后直接提取组织RNA,还是经模拟LCM环境再提取RNA,均显示锌固定法对RNA完整性提供了良好保护。快速免疫组化可以明确标记血管内皮细胞,后者能够被LCM准确捕获,并经RT-PCR验证为高纯度的血管内皮细胞。结论快速免疫组化联合LCM技术可以从肿瘤组织原位获取高纯度血管内皮细胞,并保证RNA的完整性,可能为肿瘤血管内皮细胞基因表达研究奠定基础。

Objective To develope a feasible approach of obtaining tumor vascular endothelial cells of high purity from in situ tumor tissues.Methods Clinical specimens were fixed in the zinc fixation fluid before RNA extraction, and the integrity of RNA was checked by gel electrophoresis to determine the protective effect of the fluid. Laser capture micro-dissection (LCM) condition was also simulated to examine the protective effect of the fluid for the LCM process. The in situ tumor vascular endothelial cells were pr...