人泪腺腺样囊性癌肿瘤干细胞分离培养及特征研究

The cultivation and identification of lacrimal gland adenoid cystic cancer stem cells

目的 分离、培养人泪腺腺样囊性癌（LACC）细胞系并研究其肿瘤干细胞（CSC）特性.方法 实验研究.应用无血清悬浮培养法分离培养人LACC-CSC,显微镜下观察细胞形态学改变.实验分为两组,即LACC-CSC实验组和LACC对照组,应用流式细胞仪检测干细胞标记CD133、ATP结合转运蛋白G超家族成员2（ABCG2）的表达,Tanswell小室检测CSC侵袭力,并诱导CSC向血管内皮细胞分化,体外异种移植检测CSC致瘤力.实验组与对照组组间比较采用t检验.结果 LACC接种于无血清培养基后悬浮成球生长,10～ 12 d可形成形态规则的肿瘤微球体.流式细胞仪检测示LACC-CSC表达CD133比率为（35.67±6.86）％,LACC为（0.46±0.48）％,二者比较差异具有统计学意义（t=-8.867,P＜0.05）;LACC-CSC表达ABCG2为（39.99±4.54）％,LACC为（6.75±1.34）％,二者比较差异具有统计学意义（t=-9.932, P＜0.05）.Transwell体外侵袭实验示,培养24 h后,LACC-CSC穿过Matrigel基底膜平均为（32.60±8.79）个/高倍镜视野,LACC为（10.20±2.77）个/高倍镜视野,两组比较差异有统计学意义（t=5.433,P＜0.05）;培养48 h后LACC-CSC穿过Matrigel基底膜平均为（62.60±4.83）个/HP,LACC为（44.00±5.34）个/HP,两组比较差异有统计学意义（t=5.779,P＜0.05）.应用含VEGF和bFGF等的血清培养基低氧诱导,LACC-CSC呈贴壁生长,细胞形态改变,连续诱导后三维基质胶中可形成血管腔样结构,表达血管内皮标记CD31、CD34.体外移植瘤实验LACC-CSC组9d时全部出瘤,LACC组12d全部出瘤,两组成瘤率均为100％.结论 通过无血清培养法获得的LACC-CSC悬浮成球生长,表达经典干细胞标记CD133及ABCG2,具有更强的迁移侵袭力以及体内致瘤力,并具有多向分化潜能,具有干细胞的一般特性.

Objective To isolate and cultivate the Lacrimal gland Adenoid Cystic Carcinoma cells line, study Cancer Stem Cells properties.Method Experimental study.Lacrimal gland adenoid cystic carcinoma cancer stem cells were cultivated in serum-free suspension culture and the morphological changes were observed.Cells were divided into two groups,the LACC-CSC experimental group and the LACC control group.The flow cytometry instrument was used to detect the expression of classical stem cell markers CD 133 and ABCG2.Transwell chamber was used to detect the cancer stem cell aggressivity and differentiated into the vascular endothelial cells.The tumorigenic force in vitro xenotransplantation were applied.Result LACC cells can grow suspensively after vaccinated in serum free medium and form tumor microspheres after 10-12 days.Flow cytometry experiments showed that the expression ratio of stem cell markers CD133 in LACC-CSC was （35.67± 6.86）%, significantly different to LACC with （0.46 ±0.48）%, （t=8.867, P〈0.05）.Similarly ,the expression ratio of stem cell marker ABCG2 in LACC-CSC was （39.99±4.54）%, significantly different to LACC with （6.75 ± 1.34）%,（t=-9.932, P〈0.05）.In vitro experiment of Matrigel invasion, LACC-CSC went through the matrigel basement membrane averagely （32.60± 8.79）/HP contrary to LACC with average （10.20±2.77）/HP after 24 hours, showing statistically significance（t=5.433, P〈0.05） between the two groups.After training for 48 hours, the difference between two groups was still obvious （t=5.779, P〈0.05） with LACC-CSC average（62.60±4.83）/HP to LACC （44.00±5.34）/HP.When induced by serum medium containing VEGF and bFGF, LACC-CSC grew adherent gradually and cell morphological changes occurred after continuous induction to long spindle cells.When cultured into three-dimensional matrix structure they formed vessel samples and expressed vascular endothelial marker CD31 and CD34.Transplanted tumor in vitro experiment, mice of LACC-CSC group grew tumors in 9 days with 100% tumorigenic rate, whereas LACC group 12 days with 100% tumorigenic rate.Conclusions LACC-CSC can be obtained through serum-free culture method.LACC-CSC grew suspensively and expressed classical stem cell markers.LACC-CSC were identified as cancer stem cells with stronger migration and invasion.LACC-CSC have tumorigenic force and multi-directional differentiation potential with general characteristics of the stem cell.