中文摘要：

目的研究人乳腺癌细胞MCF-7获得他莫昔芬（tamoxifen,TAM）耐药过程中发生的肌动蛋白细胞骨架重构及其对细胞迁移能力的影响,并探讨相关分子机制。方法采用高浓度短时间4-羟基他莫昔芬（4-hydroxytamoxifen,OHT）冲击法诱导人乳腺癌MCF-7/TAM耐药细胞株（Tam-R）。运用FITC标记的鬼笔环肽染色观察纤维状肌动蛋白（F-actin）动态变化,免疫荧光分析E-钙粘蛋白在野生型MCF-7细胞（MCF-7W）及Tam-R细胞中的表达及分布,pull-down和Westernblot检测小GTP酶Rac1活性,Transwell细胞迁移实验评估F-actin骨架重构对Tam-R细胞迁移能力的影响。结果 MCF-7W细胞中F-actin富集于毗邻细胞膜周边,呈典型鹅卵石形态,E-钙粘蛋白分布与F-actin相似,可在毗邻细胞膜周边形成完整的黏附连接;而Tam-R细胞中F-actin纤维出现板状伪足和应力纤维两种异常形态,细胞外围不能通过E-cadherin与周围细胞形成完整的黏附连接。在Tam-R细胞中,PI3K抑制剂Wortmannin（WM）可抑制OHT引起的F-actin骨架重构、Rac1的活化和细胞迁移（P〈0.05）,而ERK1/2抑制剂U0126对OHT引起的F-actin骨架重构无明显影响。结论 OHT可能激活PI3K,促进Rac1活化,通过诱导F-actin骨架重构促进Tam-R细胞迁移。

英文摘要：

Objective To investigate the rearrangement of filament actin（F-actin） cytoskeleton during the development of tamoxifen resistance in human breast cancer MCF-7 cells. Methods MCF-7 tamoxifen-re- sistant （Tam-R） cells were derived from wild-type MCF-7 （MCF-7W） cells by exposure to a high concentration of 4-hydroxytamoxifen （OHT） for a short period. The dynamic change of F-actin was visualized by FITC-Phal- loidin staining. Immunofluoreseence staining was used to evaluate the expression and distribution of E-cadherin in MCF-7W and Tam-R cells. Pull-down assay small GTPases Racl. Transwell assay was used and Western blot analysis were utilized to analyze the activity of to evaluate the effects of F-actin cytoskeleton rearrangement on migratory ability of Tam-R cells. Results In MCF-7W cells, F-actin concentrated along the cell membrane like pebbles, and E-cadherin, distributed like F-actin, formed strong intercellular adhesion junction. In con- trast, abnormal lamellipodia, stress fiber and reduced E-cadherin-mediated cell-cell adhesion were observed in Tam-R cells. Additionally, the PI3K inhibitor Wortmannin （WM） attenuated the activity of Racl, rearrange- ment of actin cytoskeleton and cell migration （P 〈 0. 05 ） induced by tamoxifen in Tam-R cells. The inhibitor of ERK, U0126, had few effects on the actin cytoskeleton rearrangement induced by OHT. Conclusion OHT probably activates PI3K and promotes Racl activation, and to promote the migration in Tam-R cells by inducing the rearrangement of actin cytoskeleton.