人肝癌组织原代巨噬细胞的提取与鉴定

Extraction and identification of primary macrophages from human hepatocellular carcinoma

目的研究人肝癌组织原代肿瘤相关性巨噬细胞(TAMs)的提取与鉴定方法。方法收集原发性肝癌患者术中切取的肝癌组织标本15份,免疫组织化学染色方法检测肝癌组织中肿瘤相关巨噬细胞表面标志物CD68及CD206的表达。利用机械法和酶消化法结合分离TAMs,贴壁培养24h后在倒置显微镜下进行细胞形态学观察,利用CD68和CD206作为标志物,流式细胞术鉴定细胞纯度。结果癌巢和癌旁组织中均可见CD68、CD206阳性表达,阳性细胞表现为棕褐色;获得一定纯度的原代巨噬细胞,具备巨噬细胞的形态特征,其中CD68单阳性比例为36.29%,CD68和CD206双阳性比例为15.96%。结论本研究采用的方法可有效提取和培养人肝癌组织来源的原代TAMs,对进一步研究人类肝癌组织TAMs特性及对肿瘤生物学行为的影响具有重要价值。

Objective To study the extraction and identification of primary tumor-associated macrophages (TAMs) from human hepatocellular carcinoma (HCC).Methods Fifteen hepatocellular carcinoma specimens were collected from patients with primary HCC.Immunohistochemical staining was used to detect the expression of tumor-related macrophage surface markers CD68 and CD206 in HCC tissues.TAMs were separated by mechanical method and enzymatic digestion method.After 24 hours of adherent culture,the cell morphology was observed under inverted microscope.CD68 and CD206 were used as markers to identify the purity of TAMs by flow cytometry.Results CD68 and CD206 were positive in the central and adjacent tissues,and the positive cells were brown.A certain purity primary macrophages were obtained with morphological characteristics of macrophages.The single positive rate of CD68 was 36.29%, and the double positive rate of CD68 and CD206 was 15.96%.Conclusion The method used in this study can effectively extract and cultivate primary TAMs from human HCC tissues,which is of great value to further study the characteristics of TAMs and their effects on biological behavior of tumors.