CAJ | 학술논문

目的分选肝癌细胞株SMMC-7721中的侧群(SP)细胞,探讨其体外侵袭性与上皮-间充质转化(EMT)的关系.方法采用流式细胞荧光激活分选(FACS)技术将SMMC-7721细胞分为SP细胞和非侧群(NSP)细胞两个亚群,以Transwell法检测SP细胞和NSP细胞侵袭能力,实时定量逆转录聚合酶链反应(RT-qPCR)和Western blot技术对两个亚群细胞波形蛋白(Vimentin)、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)和Snail的表达进行分析.结果 Transwell侵袭实验中SP细胞穿膜细胞数(66.3±7.4)个明显高于NSP细胞穿膜细胞数(27.5±6.5)个(P＜0.01)；SP细胞Vimentin、N-cadherin和Snail mRNA的表达水平分别是NSP细胞的9.527、6.834、8.173倍,NSP细胞E-cadherin mRNA的表达水平是SP细胞的5.353倍,差异均有统计学意义(P＜0.01)；E-cadherin、Vimentin、N-cadherin和Snail蛋白SP细胞和NSP细胞中的表达差异有统计学意义(0.174±0.014和0.935±0.012、1.117±0.012和0.314±0.011、0.975±0.017和0.179±0.013、0.917±0.014和0.202±0.013,P＜0.01).结论肝癌SMMC-7721细胞中SP细胞的体外高侵袭性可能与EMT有关.
목적 분선간암세포주SMMC-7721중적측군(SP)세포,탐토기체외침습성여상피-간충질전화(EMT)적관계.방법 채용류식세포형광격활분선(FACS)기술장SMMC-7721세포분위SP세포화비측군(NSP)세포량개아군,이Transwell법검측SP세포화NSP세포침습능력,실시정량역전록취합매련반응(RT-qPCR)화Western blot기술대량개아군세포파형단백(Vimentin)、E-개점단백(E-cadherin)、N-개점단백(N-cadherin)화Snail적표체진행분석.결과 Transwell침습실험중SP세포천막세포수(66.3±7.4)개명현고우NSP세포천막세포수(27.5±6.5)개(P＜0.01)；SP세포Vimentin、N-cadherin화Snail mRNA적표체수평분별시NSP세포적9.527、6.834、8.173배,NSP세포E-cadherin mRNA적표체수평시SP세포적5.353배,차이균유통계학의의(P＜0.01)；E-cadherin、Vimentin、N-cadherin화Snail단백SP세포화NSP세포중적표체차이유통계학의의(0.174±0.014화0.935±0.012、1.117±0.012화0.314±0.011、0.975±0.017화0.179±0.013、0.917±0.014화0.202±0.013,P＜0.01).결론 간암SMMC-7721세포중SP세포적체외고침습성가능여EMT유관.
Objective To study the in vitro invasiveness of side population (SP) cells purified from hepatoma SMMC-7721 cells and the expression of epithelial-mesenchymal transition (EMT) markers of SP cells and non-SP (NSP) cells,and to explore the relationship between invasiveness of SP cells and EMT.Methods Fluorescence-activated cell sorting (FACS) was used to sort SP cells and non-SP (NSP) cells from SMMC-7721 cell line.The in vitro invasiveness of both subpopulations was tested by using Transwell chamber assay.Quantitative real-time reverse transcriptase-pelymerase chain reaction (RT-qPCR)and Western blotting were applied to evaluate the expression of Vimentin,E-cadherin,N-cadherin and Snail in SP cells and NSP cells,respectively.Results Transwell invasion assay revealed the number of SP cells (66.3 ±7.4) penetrating the basilar membrane was significantly greater than that of NSP cells (27.5 ±6.5)(P ＜0.01).The mRNA expression levels of Vimentin,N-cadherin and Snail were significantly increased in SP cells (9.527-fold,6.834-fold and g.173-fold,respectively) as compared with those in NSP cells (P ＜ 0.01).In contrast,the decrease of E-cadherin mRNA expression in SP cells (5.353-fold) was markedly higher than that in NSP cells (P ＜ 0.01).The protein expression levels of E-cadherin,Vimentin,N-cadherin and Snail in SP cells were markedly different from those in NSP cells (0.174 ±0.014 and 0.935 ±0.012; 1.117 ±0.012 and 0.314 ±0.011; 0.975 ±0.017 and 0.179 ±0.013; 0.917 ±0.014and 0.202 ± 0.013 ; P ＜ 0.01).Conclusion SP cells from hepatoma SMMC-7721 cells have high invasiveness in vitro,which may be related to the changes in the expression of EMT markers.