13例颅咽管瘤细胞体外培养及鉴定

Cell culture and identification of craniopharyngioma in vitro: report of 13 cases

目的探讨牙釉质上皮型颅咽管瘤(CP)细胞原代培养及传代方法,检测体外培养CP细胞的生长及生物学特性。方法对病理证实为牙釉质上皮型CP的新鲜组织标本13例,采用显微镜下组织修剪、胰酶差速贴壁消化法结合机械刮除法建立CP细胞株。用倒置显微镜、苏木精-伊红(HE)染色观察细胞生长情况及细胞形态,透射电镜(TEM)观察细胞超微结构,免疫组化鉴别细胞来源,噻唑蓝(MTT)比色法绘制细胞增殖曲线,并用流式细胞仪(FCM)分析细胞周期。结果 13例CP组织分离纯化并原代培养成功9例(69.23%)、传代培养成功7例(53.85%),最长传7代,存活63 d。广谱角蛋白免疫组织化学鉴定CP细胞纯化良好。第2代CP细胞比第6代生长稳定、存活时间长。第2代CP细胞为典型上皮样形态及二倍体细胞,细胞周期主要集中G0-G1和G2-M期。结论牙釉质上皮型CP细胞原代纯化培养获得成功,可进行有限地连续传代培养,其存活时间及生长状态可满足进一步处理实验要求。

Objective An efficient method of primary culture and subculture of adamantinomatous craniopharyngioma （CP） cells is discussed and the growth and biological characteristics of cells in vitro are investigated. Methods All 13 cases of adamantinomatous CP fresh tissue samples were confirmed by pathology. CP cell line was established by pruning under microscope, and digesting cells were examined by pancreatic enzyme at different speed adherence and mechanical shaving. The cell growth and merphology were observed by inverted microscope and hematoxylin-eosin （HE） staining. The cell ultrastructure was observed by transmission electron microscope （TEM）. The expression of pan keratin （Pan-Ck） was examined by immunocytochemistry. Methl thiazolyl tetrazolium （MTF） assay was used to draw the cell growth curve and flow cytometer （FCM） to analyze the cell cycle. Results In 13 cases of CP tissues purified, 9 cases of original generation were cultured successfully （69. 23% ）, and 7 cases were subcultured successfully （53.85%）. The oldest cells survived for 7 generations and 63 d. The second generation of CP cells grew more stable and survived longer time than the sixth generation. The characteristics of second generation CP cells were typical epithelioid cells and diploid cells, with cell cycle mainly in G0-G1 and G2 - M period. Conclusion Adamantinomatous CP cells of primary purification are cultured successfully and can continuously subcultured with limitation. Its survival and growth characteristics can meet the requirements of further experiment.