【作者】 张莹；

【导师】 张兵兵； 吕永钢；

【作者基本信息】 重庆大学 ， 生物学， 2017， 硕士

【摘要】 肿瘤转移是恶性肿瘤的重要生物学特征,大多数肿瘤患者并非死于原发性癌而是死于转移性癌。近年来,针对循环肿瘤细胞(circulating tumor cells,CTCs)外渗机制的研究已成为抑制肿瘤转移的新热点。本课题采用悬浮培养的方法模拟CTCs在血液循环系统中的悬浮力学状态,探讨悬浮力学状态对乳腺肿瘤细胞与内皮细胞之间相互作用的影响,重点考察了细胞粘附分子(cell adhesion molecules,CAMs)整合素β1(integrinβ1)在该过程中所扮演的角色。主要研究工作和实验结果如下:(1)悬浮培养促进乳腺肿瘤细胞的外渗通过流动腔粘附实验和Transwell细胞迁移实验检测贴壁组和悬浮组MDA-MB-231乳腺肿瘤细胞在内皮细胞层的粘附能力、铺展能力及跨内皮迁移能力。流动腔粘附实验结果表明:经悬浮培养1天或3天的MDA-MB-231细胞,其在内皮细胞层的粘附能力显著强于贴壁组(P<0.05),悬浮培养1天和3天之间无显著性差异。利用软件ImageJ对乳腺肿瘤细胞的铺展形态进行统计分析,结果表明:在相同共孵育时间下,悬浮组MDA-MB-231细胞在内皮细胞层的铺展面积显著大于贴壁组(P<0.01);共孵育6小时,悬浮组MDA-MB-231细胞圆度显著小于贴壁组。Transwell细胞迁移实验结果表明:悬浮组MDA-MB-231细胞发生跨内皮迁移的细胞数量显著多于贴壁组(P<0.01)。(2)悬浮力学状态诱导整合素β1高表达通过qPCR和免疫印迹法检测贴壁组和悬浮组MDA-MB-231细胞整合素β1的表达水平。实验结果表明悬浮培养使得整合素β1在基因水平的表达量提高了2.35±0.58倍(P<0.05),在蛋白水平的表达量提高了2.17±0.40倍(P<0.01)。通过siRNA技术对整合素β1进行敲低处理,qPCR结果表明:贴壁组MDA-MB-231细胞的整合素β1在基因水平上降低了62%(贴壁对照组化1处理,P<0.01),敲低效果极显著;悬浮组MDA-MB-231细胞的整合素β1在基因水平上降低了74%(P<0.01),敲低效果极显著。免疫印迹法结果表明:整合素β1敲低处理后,贴壁组MDA-MB-231细胞的整合素β1在蛋达水平上降低了39%(贴壁对照组化1处理,P<0.01),敲低效果极显著;悬浮组MDA-MB-231细胞的整合素β1在蛋白水平上降低了80%(P<0.01),敲低效果极显著。(3)整合素β1对乳腺肿瘤细胞外渗的影响设置贴壁对照组(Ad-Ctrl)、贴壁整合素β1敲低组(Ad-ITGB1)、悬浮对照组(Susp-Ctrl)、悬浮整合素β1敲低组(Susp-ITGB1),通过流动腔粘附实验、铺展实验和Transwell细胞迁移实验检测整合素β1对MDA-MB-231细胞外渗能力的影响。实验结果表明:整合素β1敲低组MDA-MB-231细胞(si-ITGB1)与阴性对照组(si-Ctrl)相比,在内皮细胞层的粘附数量显著降低;si-ITGB1组和si-Ctrl组MDA-MB-231在内皮细胞层的铺展无显著性差异;si-ITGB1组与si-Ctrl组相比,发生迁移的细胞数量显著降低。本文的研究结果表明:悬浮培养可提高MDA-MB-231乳腺肿瘤细胞在血管内皮细胞层的粘附能力、铺展能力和跨内皮迁移能力,可能的机制是悬浮力学状态诱导MDA-MB-231细胞整合素β1表达上调。相关结论对理解CTCs在转移过程中的粘附迁移有一定的参考意义。更多还原

【Abstract】 Tumor metastasis is an important biological feature of malignant tumors,most cancer patients died of metastatic carcinoma,not primary carcinoma.In recent years,the extravasation of circulating tumor cells has became one of the hot points of inhibiting tumor metastasis.In this study,the suspension state of CTCs was mimicked to explore it’s effect on extravasation of breast tumor cells.The role of cell adhesion molecule-integrin β1 in this process was also explored.The main works and results are as follows:(1)Suspension culture promote extravasation of breast tumor cells.The ability of adhesion,spreading and transendothelial migration of adherent and suspended breast cancer cells were evaluated by parallel-plate flow chamber adhesion assay,spreading assays and transwell assay.The results of parallel-plate flow chamber adhesion assay showed that the adhesion ability of suspended MDA-MB-231 cells is stronger than adherent ones(P < 0.05).There is no significantly difference between the suspension culture for 1 day and 3 days.Quantification of the areas and circularities of MDA-MB-231 cells spreading on endothelial cells monolayer,the results showed that the spreading ability of suspended MDA-MB-231 cells is stronger than adherent ones(P < 0.01).The results of Transwell assay showed that the transendothelial migration ability of suspended MDA-MB-231 cells is stronger than adherent ones(P < 0.01).(2)Suspension state promote the expression of integrin β1.Integrin β1’s expression levels of adherent and suspended MDA-MB-231 cells were detected by qPCR and western blot.The qPCR results showed that integrin β1’s expression level of suspended MDA-MB-231 cells is 2.35 ± 0.58 times of the adherent ones(P < 0.05).The western blot results showed that integrin β1’s expression level of suspended MDA-MB-231 cells is 2.17 ± 0.40 times of the adherent ones(P < 0.01).Knock-down of integrin β1 significantly decreased 62% of ITGB1 mRNA level in adherent MDA-MB-231 cells and 74% of ITGB1 mRNA level in suspended MDA-MB-231 cells.Knock-down of integrin β1 significantly decreased 39% of integrin β1 protein level in adherent MDA-MB-231 cells and 80% of integrin β1 protein level in suspended MDA-MB-231 cells.(3)The effect of integrin β1 on extravasation of breast cancer cells.The ability of adhesion,spreading and transendothelial migration of Ad-ctrl,Ad-ITGB1,Susp-ctrl,Susp-ITGB1 breast cancer cells were evaluated by parallel-plate flow chamber assay spreading assay and transwell assay.The experimental results showed that the integrin β1 have an effect both on adhesion and transendothelial migration of tumor cells,but have no influence on cell spreading.In summary,the suspension state inducing integrin β1 high expression promoted extravasation of breast tumor cells.The possible mechanism is that the suspension state promoted extravasation of breast tumor cells by inducing integrin β1 high expression.The results have some value for the understanding of tumor metastasis.更多还原