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
란소상피성암림파결고전이세포화제정맥내피세포교호화공동배양후세포특정적변화
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
  • 万方数据
    中华妇产科杂志 중화부산과잡지
    CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
    2014年 7期 510-516 ,共7页

    卢迎新%高婷%陈艳华%张新颖%李力%谢一泓%李鸿%阮和云%黎丹戎

    로영신%고정%진염화%장신영%리력%사일홍%리홍%원화운%려단융

    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 卵巢腫瘤%淋巴轉移%腫瘤細胞,培養的%人臍靜脈血管內皮細胞%共同培養技術%培養基,條件性
    란소종류%림파전이%종류세포,배양적%인제정맥혈관내피세포%공동배양기술%배양기,조건성
    Ovarian neoplasms%Lymphatic metastasis%Tumor cells,cultured%Human umbilical vein endothelial cell%Coculture techniques%Culture medis,conditioned
    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/PM4+HUVEC细胞中高表达，SKOV3/PM4+HUVEC细胞、SKOV3/PM4细胞中MMP-2的灰度值分别为1885±84和1209±114，两者比较，差异有统计学意义（P<0.05）；而MMP-9在HUVEC细胞、SKOV3/PM4细胞、SKOV3/PM4+HUVEC细胞均不表达。结论 SKOV3/PM4、HUVEC细胞交互培养、共同培养分别与单独培养相比，其细胞特征均发生明显改变，推测可能与细胞生长的微环境及细胞间相互作用有关。
    목적：탐토란소상피성암（란소암）림파결고전이세포화제정맥내피세포체외교호화공동배양후세포특정적변화。방법건립록색형광단백（GFP）표기적란소암림파결고전이세포주SKOV3/PM4세포화세포막홍색형광염료DiI표기적인제정맥내피세포주HUVEC세포，분별수집SKOV3/PM4、HUVEC세포적배양상청액，작위교호배양적조건배양기（여이HUVEC세포배양적상청액배양SKOV3/PM4세포），건립량충세포교호배양화공동배양체계。세포교호배양후，광경관찰세포형태적변화병계산세포분렬지수，투사전경관찰세포초미결구적변화，사갑기우담서람（MTT）비색법검측세포생장속도，류식세포의분석세포주기비례；세포공동배양후，격광공취초현미경관찰량충세포간적상호작용정황，명효매보법검측세포중기질금속단백매2（MMP-2）화MMP-9적표체。결과세포교호배양후，여단독배양적SKOV3/PM4세포상비，교호배양적SKOV3/PM4세포위족증다，핵분렬상[세포분렬지수분별위（4.8±0.8）%、（11.2±0.3）%；P<0.05]증다，생장속도명현증쾌，G0/G1기세포비례[분별위（69.4±3.6）%、（48.4±4.6）%；P<0.05]강저，G2/M기세포비례[분별위（5.2±1.6）%、（24.9±2.2）%；P<0.05]승고；여단독배양적HUVEC세포상비，교호배양적HUVEC세포형태개변명현，출현공포화초미결구，핵분렬상[세포분렬지수분별위（2.7±0.5）%、（5.7±0.6）%；P<0.05]증다，생장속도략강저，G0/G1기세포비례[분별위（51.4±2.2）%、（79.0±4.1）%；P<0.05]승고，G2/M기세포비례[분별위（19.1±1.2）%、（3.3±0.5）%；P<0.05]강저。세포공동배양48 h후，격광공취초현미경관찰，록색형광표기적SKOV3/PM4세포화홍색형광표기적HUVEC세포출현융합현상；명효매보법검측현시，MMP-2재HUVEC세포중불표체，재SKOV3/PM4세포중저표체，재공동배양적SKOV3/PM4+HUVEC세포중고표체，SKOV3/PM4+HUVEC세포、SKOV3/PM4세포중MMP-2적회도치분별위1885±84화1209±114，량자비교，차이유통계학의의（P<0.05）；이MMP-9재HUVEC세포、SKOV3/PM4세포、SKOV3/PM4+HUVEC세포균불표체。결론 SKOV3/PM4、HUVEC세포교호배양、공동배양분별여단독배양상비，기세포특정균발생명현개변，추측가능여세포생장적미배경급세포간상호작용유관。
    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 cell cycles, it showed the raised cell ratio in G0/G1 phase, respectively [(51.4 ± 2.2)%,(79.0 ± 4.1)%;P<0.05] and the declined cell ratio in G2/M phase, respectively [(19.1±1.2)%, (3.3±0.5)%;P<0.05]. After co-culture for 48 hours, spontaneous fusion between SKOV3/PM4 and HUVEC cell was observed by the laser confocal microscope. Gelatin zymography assay showed that MMP-2 was not expressed in HUVEC cells, low-expressed in SKOV3/PM4 cells and high-expressed in the co-culture SKOV3/PM4+HUVEC cells. The expression of MMP-2 in co-culture SKOV3/PM4+HUVEC cells and SKOV3/PM4 cells respectively were 1 885 ± 84 and 1 209 ± 114 (P<0.05). But there were no MMP-9 expression in HUVEC cells, SKOV3/PM4 cells, and the co-culture SKOV3/PM4+HUVEC. Conclusion The characteristics of SKOV3/PM4 and HUVEC show significant changes after condition culture and co-culture, it may involve in the microenvironment of the cells and the intercellular crosstalk pathway.
    원문보기 원문다운로드 사이트로이동
저자의 최근 논문