肝内胆管细胞癌相关成纤维细胞的分离、纯化及鉴定

目的：探讨肝内胆管癌相关成纤维细胞（iCAFs）的分离、纯化和鉴定方法,为后续研究奠定基础。方法：在无菌条件下,留取肝内胆管细胞癌（iCCA）组织标本,充分剪碎后置入含Ⅳ型胶原酶和透明质酸酶的消化液中恒温振荡,将所得的单细胞悬液进行免疫磁珠分选纯化,分离出肿瘤相关成纤维细胞（CAFs）并培养。通过观察细胞形态,并经免疫荧光和流式细胞仪测定,鉴定其是否为iCAFs。结果：iCAFs呈长条形或纺锤形,胞质透明,折光明显,胞核单个、呈圆形或卵圆形,细胞突起相互交错重叠形成网格状排列、密集时呈现＂峰-谷＂样表现;免疫荧光检测显示,α平滑肌肌动蛋白（＋）、波形蛋白（＋）、复合型角蛋白抗体（-）;流式细胞...

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Bibliographic Details
Published in中国临床医学 Vol. 23; no. 2; pp. 131 - 135
Main Author 杨柳晓 高强
Format Journal Article
LanguageChinese
Published 复旦大学附属中山医院重症医学科,上海,200032%复旦大学附属中山医院肝外科,上海,200032 2016
Subjects
分离
肝内胆管细胞癌
肿瘤相关成纤维细胞
鉴定
carcinoma-associated fibroblasts
identification
鉴定
肝内胆管细胞癌
isolation
intrahepatic cholangiocarcinoma
肿瘤相关成纤维细胞
分离
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ISSN1008-6358

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Summary:目的：探讨肝内胆管癌相关成纤维细胞（iCAFs）的分离、纯化和鉴定方法,为后续研究奠定基础。方法：在无菌条件下,留取肝内胆管细胞癌（iCCA）组织标本,充分剪碎后置入含Ⅳ型胶原酶和透明质酸酶的消化液中恒温振荡,将所得的单细胞悬液进行免疫磁珠分选纯化,分离出肿瘤相关成纤维细胞（CAFs）并培养。通过观察细胞形态,并经免疫荧光和流式细胞仪测定,鉴定其是否为iCAFs。结果：iCAFs呈长条形或纺锤形,胞质透明,折光明显,胞核单个、呈圆形或卵圆形,细胞突起相互交错重叠形成网格状排列、密集时呈现＂峰-谷＂样表现;免疫荧光检测显示,α平滑肌肌动蛋白（＋）、波形蛋白（＋）、复合型角蛋白抗体（-）;流式细胞仪检测示,α平滑肌肌动蛋白（＋）,同时CD34和CD45均为（-）。结论：酶消化法联合免疫磁珠分选可有效分离出高纯度的iCAFs,为进一步研究iCAFs的相关功能研究提供了良好的平台。
Bibliography:31-1794/R
Objective： To explore the methods of isolation, purification, and identification of intrahepatic cholangiocarcinoma （iCCA） associated fibroblasts （iCAFs）, which could facilitate further functional and pharmacological evaluation. Methods： Under aseptic conditions the tissue specimens of iCCA were obtained, minced, and digested in the digestive medium supplemented with collagenase IV and hyaluronidase. The single-cell suspension underwent immunomagnetic separation and purification, and carcinoma-associated fibroblasts （CAFs） were separated and cultured. Through morphological observation, immunofluorescent staining and flow cytometry analysis, whether the cells were iCAFs or not. Results. Morphologically, iCAFs were elongated or fusiform, with transparent cytoplasm, obvious refraction, single round or ovoid nucleus. Cellular processes overlap to form grids and show the ＂peak-valley＂ pattern. Under immunofluorescence, the typical iCAFs showed positive staining for alpha smooth muscle aetin （a-SMA） and vi
ISSN:1008-6358