CAJ | 학술논문

目的：了解槲皮素诱导人乳腺癌MCF－7细胞凋亡的作用及其与Fas／FasL通路的相关性。方法：建立槲皮素诱导的MCF－7细胞凋亡模型。采用透射电镜观察细胞核形态变化，Annexin V－FITC／PI和JC－1荧光标记流式细胞术检测细胞凋亡率、线粒体膜电位（Δψm ）及FasL中和抗体对细胞凋亡的阻断作用。采用免疫荧光法和流式细胞术检测细胞Fas／FasL的表达。流式细胞术观察p38 MAPK抑制剂SB203580对细胞Fas／FasL表达的影响。 Western blot检测p38 MAPK和p－p38 MAPK蛋白水平的变化。结果：80．0μmol／L槲皮素处理MCF－7细胞48 h，透射电镜可见染色质浓缩及边缘化现象；处理24 h、48 h和72 h，Δψm 分别下降17．4％、44．3％和68．9％，细胞凋亡率分别为（10．2±3．3）％、（28．9±7．5）％和（39．2±8．9）％。 FasL中和抗体预处理细胞后，24 h、48 h和72 h的细胞凋亡率则分别为（8．2±2．8）％、（19．2±5．3）％和（22．5±6．9）％，细胞凋亡阻断率分别为19．6％、33．6％和42．6％。 Fas／FasL表达率随处理时间增加而升高，SB203580可显著抑制细胞Fas／FasL的表达率。 p38 MAPK的蛋白水平变化不大，而p－p38 MAPK的蛋白水平在48 h和72 h显著增高。结论：槲皮素可上调MCF－7细胞的Fas／FasL表达，并经膜Fas／FasL途径诱导MCF－7细胞凋亡，p－p38 MAPK可能是上调Fas／FasL表达的重要信号分子。
목적：료해곡피소유도인유선암MCF－7세포조망적작용급기여Fas／FasL통로적상관성。방법：건립곡피소유도적MCF－7세포조망모형。채용투사전경관찰세포핵형태변화，Annexin V－FITC／PI화JC－1형광표기류식세포술검측세포조망솔、선립체막전위（Δψm ）급FasL중화항체대세포조망적조단작용。채용면역형광법화류식세포술검측세포Fas／FasL적표체。류식세포술관찰p38 MAPK억제제SB203580대세포Fas／FasL표체적영향。 Western blot검측p38 MAPK화p－p38 MAPK단백수평적변화。결과：80．0μmol／L곡피소처리MCF－7세포48 h，투사전경가견염색질농축급변연화현상；처리24 h、48 h화72 h，Δψm 분별하강17．4％、44．3％화68．9％，세포조망솔분별위（10．2±3．3）％、（28．9±7．5）％화（39．2±8．9）％。 FasL중화항체예처리세포후，24 h、48 h화72 h적세포조망솔칙분별위（8．2±2．8）％、（19．2±5．3）％화（22．5±6．9）％，세포조망조단솔분별위19．6％、33．6％화42．6％。 Fas／FasL표체솔수처리시간증가이승고，SB203580가현저억제세포Fas／FasL적표체솔。 p38 MAPK적단백수평변화불대，이p－p38 MAPK적단백수평재48 h화72 h현저증고。결론：곡피소가상조MCF－7세포적Fas／FasL표체，병경막Fas／FasL도경유도MCF－7세포조망，p－p38 MAPK가능시상조Fas／FasL표체적중요신호분자。
[ABSTRACT]AIM:ToinvestigatetheroleofquercetinintheapoptosisofhumanbreastcancercelllineMCF-7 and the association with Fas/Fas ligand (FasL) pathway.METHODS: The apoptosis model of MCF-7 cells was estab-lished by the induction with quercetin .The morphological characteristics of apoptotic MCF-7 cells were observed under transmission electron microscope .The apoptotic rates and alternation of mitochondrial membrane potential (Δψm ) in the MCF-7 cells were measured by flow cytometry using fluorescein labeled Annexin V-FITC/PI and JC-1, respectively.FasL neutralizing antibody was applied to block the apoptosis .The expression of Fas/FasL on the cells was detected by immuno-fluorescence technique and flow cytometry , respectively.The influence of SB203580 (an inhibitor of p38 MAPK) on the expression of Fas/FasL was also examined by flow cytometry .The protein levels of p 38 MAPK and p-p38 MAPK were de-termined by Western blot .RESULTS: The phenomenon of nuclear condensation and marginalization in the MCF -7 cells treated with quercetin at 80.0 μmol/L for 48 h was observed under transmission electron microscope .Compared with the control cells , theΔψm was decreased by 17.4%, 44.3% and 68.9% in the MCF-7 cells treated with quercetin at 80.0μmol/L for 24 h, 48 h and 72 h, respectively .The apoptotic rates of MCF-7 cells treated with quercetin at 80.0 μmol/L for 24 h, 48 h and 72 h were (10.2 ±3.3)%, (28.9 ±7.5)%and (39.2 ±8.9)%, respectively.However, the apop-totic rates were decreased to (8.2 ±2.8)%, (19.2 ±5.3)% and (22.5 ±6.9)% after the cells were pretreated with FasL neutralizing antibody , respectively .When MCF-7 cells were treated with quercetin for 24 h, 48 h and 72 h, Fas/FasL expression rates were increased in a time-dependent manner , which were largely inhibited by SB203580.The protein level of p38 MAPK was not changed obviously , but the protein level of p-p38 MAPK was significantly increased at 48 h and 72 h.CONCLUSION: Quercetin up-regulates the expression of Fas/FasL on MCF-7 cells, and induces apoptosis via Fas/FasL pathway .Meanwhile , p-p38 MAPK is potentially critical signaling molecule for up-regulating the expression of Fas/FasL.