卵巢上皮性癌淋巴结高转移细胞和脐静脉内皮细胞交互和共同培养后细胞特征的变化  

Characteristics change of the human directional highly lymphatic metastasis ovarian carcinoma cell and venous endothelial cell after establishment of their condition cultrue and co-culture cell system

作　　者：卢迎新[1] 高婷[1] 陈艳华[1] 张新颖[1] 李力[1] 谢一泓[1] 李鸿[1] 阮和云[1] 黎丹戎[1] 

机构地区：[1]广西医科大学附属肿瘤医院妇瘤科,南宁530021

出　　处：《中华妇产科杂志》2014年第7期510-516,共7页Chinese Journal of Obstetrics and Gynecology

基　　金：国家自然科学基金（81060218,81360502）;广西自然科学基金（2012GXNSFAA053157）

摘　　要：目的：探讨卵巢上皮性癌（卵巢癌）淋巴结高转移细胞和脐静脉内皮细胞体外交互和共同培养后细胞特征的变化。方法建立绿色荧光蛋白（GFP）标记的卵巢癌淋巴结高转移细胞株SKOV3/PM4细胞和细胞膜红色荧光染料DiI标记的人脐静脉内皮细胞株HUVEC细胞，分别收集SKOV3/PM4、HUVEC细胞的培养上清液，作为交互培养的条件培养基（如以HUVEC细胞培养的上清液培养SKOV3/PM4细胞），建立两种细胞交互培养和共同培养体系。细胞交互培养后，光镜观察细胞形态的变化并计算细胞分裂指数，透射电镜观察细胞超微结构的变化，四甲基偶氮唑蓝（MTT）比色法检测细胞生长速度，流式细胞仪分析细胞周期比例；细胞共同培养后，激光共聚焦显微镜观察两种细胞间的相互作用情况，明胶酶谱法检测细胞中基质金属蛋白酶2（MMP-2）和MMP-9的表达。结果细胞交互培养后，与单独培养的SKOV3/PM4细胞相比，交互培养的SKOV3/PM4细胞伪足增多，核分裂象[细胞分裂指数分别为（4.8±0.8）%、（11.2±0.3）%；P〈0.05]增多，生长速度明显增快，G0/G1期细胞比例[分别为（69.4±3.6）%、（48.4±4.6）%；P〈0.05]降低，G2/M期细胞比例[分别为（5.2±1.6）%、（24.9±2.2）%；P〈0.05]升高；与单独培养的HUVEC细胞相比，交互培养的HUVEC细胞形态改变明显，出现空泡化超微结构，核分裂象[细胞分裂指数分别为（2.7±0.5）%、（5.7±0.6）%；P〈0.05]增多，生长速度略降低，G0/G1期细胞比例[分别为（51.4±2.2）%、（79.0±4.1）%；P〈0.05]升高，G2/M期细胞比例[分别为（19.1±1.2）%、（3.3±0.5）%；P〈0.05]降低。细胞共同培养48 h后，激光共聚焦显微镜观察，绿色荧光标记的SKOV3/PM4细胞和红色荧光标记的HUVEC细胞出现融合现象；明胶酶谱法检测显示，MMP-2在HUVEC细胞中不表达，在SKOV3/PM4细胞中低表达，在共同培养的SKOV3/Objective To establish the condition cultrue cell system and co-culture cell system with SKOV3/PM4,HUVEC and to study the changes of their biological characteristics. Methods The cells of SKOV3/PM4 and HUVEC were labeled with green and red fluorescent respectively. The cell supernatant of SKOV3/PM4 and HUVEC were collected respectively as the condition medium（e.g：the cell supernatant of HUVEC cells was used as SKOV3/PM4 condition medium）and to establish the condition cultrue cell system and the co-culture cell system of the two cell lines. In the condition cultrue cell system, The morphological changes of cells were observed by HE staining to calculate the mitotic index. The ultrastructural changes of the two cells were observed by transmission electron microscopy（TEM）. The growth curve of the cells was determined by methyl thiazolyl tetrazolium （MTT） assay and flow cytometry was used to analyzed the cell cycles.In the co-culture cell system, the interaction of the two cells were detected by laser scanning confocal microscope（LSCM）. The expression of matrix metalloproteinase-2（MMP-2） and matrix metalloproteinase-9 （MMP-9） were detected by gelatin zymography. Results Compared with the single culture SKOV3/PM4, the cells which cultured in HUVEC condition medium showed the increase of pseudopodia and nuclear division,the mitotic index respectively were [（4.8 ± 0.8）%,（11.2 ± 0.3）%;P〈0.05]. The growth rate was significantly increased. In cell cycles, it showed the declined cell ratio of G0/G1 phase, respectively[（69.4±3.6）%, （48.4±4.6）%;P〈0.05] and the raised cell ratio of G2/M phase, respectively [（5.2±1.6）%, （24.9±2.2）%;P〈0.05]. Compared with the single culture HUVEC,the cells which cultured in SKOV3/PM4 condition medium showed the significant morphological change and vacuolization in the cytoplasm, Nuclear division was increased and the mitotic index respectively were [（2.7±0.5）%, （5.7±0.6）%;P〈0.05]. The growth rate was slightly declined. In

关 键 词：卵巢肿瘤 淋巴转移 肿瘤细胞 培养的 人脐静脉血管内皮细胞 共同培养技术 培养基 条件性 

分 类 号：R737.31[医药卫生—肿瘤]