人泪腺腺样囊性癌细胞的原代培养及其形态学特征

Primary culture and morphological characteristics of human lacrimal adenoid cystic carcinoma cells

目的 探讨人泪腺腺样囊性癌细胞的原代培养方法.方法 实验研究.取2011年5月16日至6月1日在天津医科大学眼科医院接受手术治疗的1位泪腺腺样囊性癌患者的术中标本,采用组织块培养法获得原代细胞.运用连续传代、机械刮除、反复贴壁和消化排除等方法去除杂细胞.通过相差显微镜、扫描电镜、透射电镜等观察细胞形态.免疫细胞化学法检测细胞波形蛋白（vimentin）、结蛋白（desmin）、S-100蛋白、细胞角蛋白（cytokeratin,pan）、CD34等蛋白的表达情况.免疫组织化学法检测肿瘤组织vimentin、desmin、S-100蛋白、cytokeratin（pan）等蛋白的表达情况.消化法绘制细胞生长曲线,计算细胞倍增时间.结果 组织块原代培养第5天见周围开始有细胞爬出,此后细胞缓慢增殖,至第32天进行首次传代,第69天发现典型的上皮样细胞克隆.运用多种方法去除杂细胞后,至第8代细胞基本纯化.纯化后细胞现已稳定传至100代以上.相差显微镜下观察第25代细胞呈典型的上皮样细胞形态,可出现接触抑制消失.扫描电镜和透射电镜下第25代细胞呈现低分化恶性肿瘤细胞的典型特点,细胞间信号沟通丰富.细胞免疫化学法检测第25代癌细胞vimentin、cytokeratin （pan）和S-100蛋白呈阳性,desmin、CD34蛋白呈阴性,与源肿瘤组织染色结果一致.细胞生长曲线类似“S”形,细胞倍增时间37.1 h.结论 采用组织块培养法可以获得基本纯化的人泪腺腺样囊性癌细胞,有可能通过此法获得稳定的人泪腺腺样囊性癌细胞系.

Objective To explore a method of primary culturing human adenoid cystic carcinoma cells of lacrimal gland.Methods Experiment research.Tumor tissue was obtained from the surgical material of a patient diagnosed as lacrimal adenoid cystic carcinoma in Tianjin Medical University Eye Hospital during May 16th to June 1st.We gained primary cells via tissue culture techniques.Mixed cells were removed through several ways.Observed cell morphological characteristics by phase contrast microscope,scanning electron microscope and transmission electron microscope.Cyto-immunochemical staining was applied to detect the expressions of vimentin,desmin,S-100,cytokeratin,pan and CD34.Their expressions were also detected in the tumor tissue except CD34.Made cell growth curve and calculated cell doubling time.Results The outgrowth of cells was observed by day 5 after seeding tissues,and then cells generated slowly.The first passage proceeded by day 32,and the classical epithelioid cell colonies was observed by day 69 after inoculation.Purified cells of human lacrimal adenoid cystic carcinoma were obtained after the removal of mixed cells through several ways,which have been successfully subcultured for more than 100 passages.The 25th passage LACC cells appeared to be typically epithelioid cells,they showed contact inhibition as the density high enough.SEM and TEM showed the 25th passage LACC cells were malignant tumor cells poorly differentiated.They showed positive reaction with vimentin、cytokeratin （pan） as well as S-100,but negative reaction with desmin and CD34,which were consistent with the tumor tissue.The cell growth curve turned like a sigmoid one,and the cell doubling time was 37.1 h.Conclusions We gained purified LACC cells,and understood the morphological characteristics,laying the foundation forthe establishment of a human lacrimal adenoid cystic carcinoma cell line.