神经营养因子受体C依赖性调节乳腺癌细胞生物学特性的研究  

Tropomyosin-related kinase C regulating the biological characteristics of breast cancer cell T47D as a dependence receptor

作　　者：张巍[1] 牛昀[1] 刘珊[1] 牛凤婷[1] 申红红[1] 王丽[1] 

机构地区：[1]天津医科大学肿瘤医院乳腺病理研究室国家肿瘤临床医学研究中心乳腺癌防治教育部重点实验室天津市肿瘤防治重点实验室,300060

出　　处：《中华实验外科杂志》2015年第5期948-953,共6页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81172532）;天津高等学校科技发展基金计划资助项目（20110126）

摘　　要：目的 探讨神经营养因子受体C（TrkC）单纯转染及TrkC和神经营养因子-3（NT-3）联合作用对乳腺癌细胞T47D生物学特性的影响及其机制.方法 在乳腺癌细胞株中筛选出不表达TrkC的T47D;以重组腺病毒Ad-TrkC转染T47D,流式细胞术和免疫荧光化学染色分析和鉴定转染效果;噻唑蓝比色法（MTT法）、平板集落形成、流式细胞仪检测癌细胞增殖与存活能力;划痕实验检测癌细胞迁移能力;Transwell和Matrigel胶检测癌细胞侵袭能力;Western blot检测相关信号分子的表达.结果 感染复数（MOI）=20重组腺病毒可成功转染T47D,转染效率达到96.7％,并使T47D成功表达TrkC;单纯TrkC转染可明显降低T47D癌细胞存活数和平板集落形成率（12.0％比35.2％,P＜0.05）,诱导癌细胞发生G2/M期阻滞,机制与癌细胞发生早期凋亡（55.5％比2.2％）和促进含半胱氨酸的天冬氨酸蛋白水解酶-3（Caspase-3）表达有关.而TrkC和NT-3的联合作用则诱导完全不同的生物学效应：促进细胞存活和平板集落形成率（84.8％,P ＜0.05）、促进细胞进入S期和G2/M期,减少细胞早期凋亡（1.8％）和死亡（P＜0.05）;机制与激活磷脂酰肌醇3激酶/蛋白激酶B（PI3K/Akt）信号通路有关,同时还能促进T47D的迁移和侵袭能力.结论 TrkC可能以依赖性受体方式调控T47D乳腺癌细胞的增殖、存活和侵袭.Objective To investigate the effects of Tropomyosin-related kinase C （neurotrophin receptor C,TrkC） re-expression in T47D with or without neurotrophin-3 （NT-3） for the biological characteristics of the cancer cell and the possible mechanisms.Methods Reverse transcription polymerase chain reaction （RT-PCR） and Western blotting were used to screen the proper cell line T47D that not expressing TrkC among four breast cancer cell lines.By recombinant adenovirus vector Ad-TrkC transfection,flow cytometry and immunofluorescence were used to evaluate the transfected efficiency.The proliferation,survival,migration and invasion of Ad-TrkC transfected T47D with or without NT-3 were estimated by thiazolyl blue tetrazolium bromide （MTF） assay,conlony-forming test,flow cytometry,wounding healing assay and Matrigel invasion assay.The related signal pathways were detected by Western blotting.Results T47D was successfully transfected by 20 multiplicity of infection （MOI） recombinant adenvirus and the tranfection efficiency arrived at 96.7%.Ad-TrkC tranfected T47D successfully expressed TrkC.The survival and the colony formation rate （12.0% vs.35.2%） were obviously suppressed （P 〈0.05） after TrkC re-expression in T47D.And TrkC induced a G2/M phase arrest in T47D.The mechanisms might be related to the more early apoptosis rate （55.5% vs.2.2%） and increased Caspase-3 expression in Ad-TrkC transfected T47D.However,combination of Ad-TrkC transfection and NT-3 induced completely different effects：increased cell survival and colony formation rate （84.8%,P 〈0.05）,more cells in S and G2/M phase and decreased cell apoptosis （1.8%） and death （P 〈 0.05）.These effects might be associated with the activation of phosphatidylinositol 3 kinase/protein kinase B （PI3K/Akt）pathway.In addition,combination of TrkC and NT-3 also promoted the migration and invasion of T47D.Conclusion TrkC may regulate the proliferation,survival and invasion of breast cancer cell T47D as a dependence receptor.

关 键 词：神经营养因子受体C 神经营养因子-3 乳腺癌 依赖性受体 增殖 凋亡 

分 类 号：R737.9[医药卫生—肿瘤]