中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2012年
4期
259-263
,共5页
刘志升%王野%李强%张圣林%史玉荣
劉誌升%王野%李彊%張聖林%史玉榮
류지승%왕야%리강%장골림%사옥영
神经节%胰腺肿瘤%神经突%细胞增殖%细胞凋亡
神經節%胰腺腫瘤%神經突%細胞增殖%細胞凋亡
신경절%이선종류%신경돌%세포증식%세포조망
Ganglia%Pancreatic neoplasms%Neurites%Cell proliferation%Apoptosis
目的 建立一种新的体外胰腺癌神经浸润( PNI)研究模型,并观察PNI过程中神经突的生长状况,以及胰腺癌细胞的增殖、凋亡和迁移情况.方法 取人胰腺癌细胞系MIA PaCa-2和大鼠背根神经节(DRG),于Matrigel胶中共培养,建立研究模型.倒置显微镜下观察胰腺癌细胞集落形成和DRG神经突生长情况以及胰腺癌细胞的迁移情况,并用Image-Pro Plus 5.0图像分析软件进行图像分析,计算神经突和胰腺癌细胞集落所占的面积.免疫细胞化学法检测胰腺癌细胞增殖细胞核抗原Ki-67的表达情况,计算胰腺癌细胞增殖指数,四甲基偶氮唑蓝(MTT)法检测胰腺癌细胞的增殖情况.利用流式细胞仪检测胰腺癌细胞的凋亡率.结果 共培养24和72 h,MIA PaCa-2/DRG共培养组的细胞集落面积分别为176.67±5.57和242.83±4.92,明显大于MIA PaCa-2细胞单独培养组(分别为151.17±5.98和182.50 ± 5.39,均P＜0.01)；MIA PaCa-2/DRG共培养组的神经突面积分别为129.33±2.73和202.50 ±8.04,明显大于DRG单独培养组(分别为63.67 ±5.17和78.67 ±3.61,均P＜0.01).神经突有明显向胰腺癌细胞集落方向生长的趋势,而神经突与胰腺癌细胞集落接触后,胰腺癌细胞则沿神经突生长的方向逆向迁移,最终到达DRG周围,出现类似PNI的表现.MTT与免疫细胞化学实验结果均表明,上清液培养组胰腺癌细胞的增殖远比普通培养基培养组活跃.流式细胞仪检测结果表明,上清液培养组胰腺癌细胞凋亡率为2.46％,明显低于普通培养基培养组(4.89％,P＜0.01).结论 MIA PaCa-2/DRG共培养模型成功构建,有助于胰腺癌PNI的研究.神经细胞与胰腺癌细胞的相互作用在胰腺癌的PNI过程中发挥重要作用.神经细胞与胰腺癌细胞可以相互促进对方的生长,并且胰腺癌细胞有沿神经突生长的方向逆向迁移的趋势.
目的 建立一種新的體外胰腺癌神經浸潤( PNI)研究模型,併觀察PNI過程中神經突的生長狀況,以及胰腺癌細胞的增殖、凋亡和遷移情況.方法 取人胰腺癌細胞繫MIA PaCa-2和大鼠揹根神經節(DRG),于Matrigel膠中共培養,建立研究模型.倒置顯微鏡下觀察胰腺癌細胞集落形成和DRG神經突生長情況以及胰腺癌細胞的遷移情況,併用Image-Pro Plus 5.0圖像分析軟件進行圖像分析,計算神經突和胰腺癌細胞集落所佔的麵積.免疫細胞化學法檢測胰腺癌細胞增殖細胞覈抗原Ki-67的錶達情況,計算胰腺癌細胞增殖指數,四甲基偶氮唑藍(MTT)法檢測胰腺癌細胞的增殖情況.利用流式細胞儀檢測胰腺癌細胞的凋亡率.結果 共培養24和72 h,MIA PaCa-2/DRG共培養組的細胞集落麵積分彆為176.67±5.57和242.83±4.92,明顯大于MIA PaCa-2細胞單獨培養組(分彆為151.17±5.98和182.50 ± 5.39,均P＜0.01)；MIA PaCa-2/DRG共培養組的神經突麵積分彆為129.33±2.73和202.50 ±8.04,明顯大于DRG單獨培養組(分彆為63.67 ±5.17和78.67 ±3.61,均P＜0.01).神經突有明顯嚮胰腺癌細胞集落方嚮生長的趨勢,而神經突與胰腺癌細胞集落接觸後,胰腺癌細胞則沿神經突生長的方嚮逆嚮遷移,最終到達DRG週圍,齣現類似PNI的錶現.MTT與免疫細胞化學實驗結果均錶明,上清液培養組胰腺癌細胞的增殖遠比普通培養基培養組活躍.流式細胞儀檢測結果錶明,上清液培養組胰腺癌細胞凋亡率為2.46％,明顯低于普通培養基培養組(4.89％,P＜0.01).結論 MIA PaCa-2/DRG共培養模型成功構建,有助于胰腺癌PNI的研究.神經細胞與胰腺癌細胞的相互作用在胰腺癌的PNI過程中髮揮重要作用.神經細胞與胰腺癌細胞可以相互促進對方的生長,併且胰腺癌細胞有沿神經突生長的方嚮逆嚮遷移的趨勢.
목적 건립일충신적체외이선암신경침윤( PNI)연구모형,병관찰PNI과정중신경돌적생장상황,이급이선암세포적증식、조망화천이정황.방법 취인이선암세포계MIA PaCa-2화대서배근신경절(DRG),우Matrigel효중공배양,건립연구모형.도치현미경하관찰이선암세포집락형성화DRG신경돌생장정황이급이선암세포적천이정황,병용Image-Pro Plus 5.0도상분석연건진행도상분석,계산신경돌화이선암세포집락소점적면적.면역세포화학법검측이선암세포증식세포핵항원Ki-67적표체정황,계산이선암세포증식지수,사갑기우담서람(MTT)법검측이선암세포적증식정황.이용류식세포의검측이선암세포적조망솔.결과 공배양24화72 h,MIA PaCa-2/DRG공배양조적세포집락면적분별위176.67±5.57화242.83±4.92,명현대우MIA PaCa-2세포단독배양조(분별위151.17±5.98화182.50 ± 5.39,균P＜0.01)；MIA PaCa-2/DRG공배양조적신경돌면적분별위129.33±2.73화202.50 ±8.04,명현대우DRG단독배양조(분별위63.67 ±5.17화78.67 ±3.61,균P＜0.01).신경돌유명현향이선암세포집락방향생장적추세,이신경돌여이선암세포집락접촉후,이선암세포칙연신경돌생장적방향역향천이,최종도체DRG주위,출현유사PNI적표현.MTT여면역세포화학실험결과균표명,상청액배양조이선암세포적증식원비보통배양기배양조활약.류식세포의검측결과표명,상청액배양조이선암세포조망솔위2.46％,명현저우보통배양기배양조(4.89％,P＜0.01).결론 MIA PaCa-2/DRG공배양모형성공구건,유조우이선암PNI적연구.신경세포여이선암세포적상호작용재이선암적PNI과정중발휘중요작용.신경세포여이선암세포가이상호촉진대방적생장,병차이선암세포유연신경돌생장적방향역향천이적추세.
Objective To establish an in vitro model of perineural invasion PNI) with co-culture of human pancreatic cancer cells and rat root ganglion,to observe the neurite outgrowth and pancreatic cancer cell proliferation and migration,and to explore the molecular basis of perineural invasion (PNI) of pancreatic cancer.Methods Human pancreatic cancer cell line ( MIA PaCa-2) and rat dorsal root ganglion (DRG) were co-cultured in Matrigel matrix to generate the PNI model.The neurite outgrowth,pancreatic cancer cell colony formation,neurite-colony contact and retrograde migration were observed under an inverted microscope.The data were analyzed with the Image-Pro Plus 5.0 system.The proliferative index (PI) was measured by immunohistochemical staining with the Ki-67 antibody.In order to determine the absorbance (A) of the pancreatic cancer cells,MTT assay was used.The apoptotic index (AI) was evaluated by flow cytometry.Results Neurite outgrowth was stimulated in the presence of pancreatic cancer cells.After 72hours of the co-culture,MIA PaCa colonies co-cultured with DRG exhibited a significantly larger colony area (242.83±4.92) than that of the control (182.50 ± 5.39,P ＜ 0.001 ).In the MIA PaCa-2/DRG coculture system,the neurites exhibited a trend of growing towards the pancreatic cancer cell colony.However,the pancreatic cancer cells showed a trend of retrogradely migrating to the DRG along the neurite outgrowth,when MIA PaCa-2 colonies touched the DRG.The positive rate of Ki-67 nuclear antigen was significantly higher than in the co-culture group.The PI value was higher in the experimental group ( 12.80％ ) than that in the control group (6.81％,P ＜0.01 ).The MTT assay showed that proliferation of the pancreatic cancer cells was more active than that in the control group.Flow cytometry analysis showed that the apoptosis rate of the pancreatic cancer cell was 2.46％,significantly lower than that of the control group (4.89％,P ＜0.001 ).Conclusions An in vitro co-culture model of rat dorsal root ganglion and human pancreatic cancer cell line is successfully established in this study.This MIA PaCa-2/DRG co-culture system demonstrates that the neural-pancreatic carcinoma cell interaction is a mutually beneficial process for the growth of neurites and pancreatic carcinoma cells. The pancreatic cancer cells show a trend of migrating to the DRG along the neurite outgrowth.
