微流控芯片对乳腺癌细胞MDA-MB-231的捕获及再培养研究

The Development of Microfluidic Chip for the Capture of Breast Cancer Cells and Its Effect on Captured Cells

目的:利用实验室构建的微流控芯片对乳腺癌细胞(MDA-MB-231)进行捕获,提高捕获率并保证细胞活性,实现再培养。抗肿瘤药物阿霉素处理正常培养和再培养的细胞,分析细胞内的基因表达变化。方法:对微流控芯片进行基底修饰,利用MUC1抗原与抗体特异性结合捕获肿瘤细胞,优化捕获条件提高捕获率。对微流控芯片捕获的细胞进行分离、收集和再培养。用1μmol/L阿霉素对正常培养和再培养的细胞分别孵育24h,然后提取RNA并逆转录合成c DNA。选择乳腺癌细胞中高表达及与肿瘤转移相关的基因FN1、ITGA6和LAMB3设计引物,以c DNA为模板分别进行RT-PCR扩增,对琼脂糖凝胶电泳结果进行灰度分析。结果:经MUC1抗体修饰的微流控芯片能有效地捕获肿瘤细胞,捕获率达80%±3%,释放率约98%,细胞释放后存活率高实现再培养。阿霉素对正常培养和再培养的乳腺癌细胞中FN1、ITGA6和LAMB3的基因表达均有抑制作用。结论:MUC1抗体修饰的微流控芯片能有效捕获乳腺癌细胞并实现再培养,捕获前后细胞内基因表达无显著差异,均能产生药物敏感性。

Objective： To achieve an effective capture efficiency of breast cancer cell（ MDA- MB-231） by the microfluidic chip,and then analyse the changes of the target genes FN1,ITGA6 and LAMB3 expressing level in the tumor cells. Methods： The substrate of microfluidic chip was coupled with MUC1 antibodies,which could capture the tumor cells by the antigen on the cell surface. The conditions of cell capture were optimized to gain high cell capture efficiency. The captured cells were released by trypsin digestion and then the released cells were collected and re-cultured. The normal and re-cultivated cells were incubated with 1μmol / L doxorubicin for 24 h respectively. Then the RNA in the cells was extracted and reversed to synthesize DNA and the target genes FN1,ITGA6 and LAMB3 were amplified by reverse transcriptase polymerase chain reaction（ RT-PCR）. Results： The tumor cells can be effectively captured by the microfluidic bio-chip after MUC1 antibodies were modified on the surface of chip and the capture rate can reach 80%. The cells release efficiency was as high as 98% and the released cells still had high viability which could be re-cultured. Doxorubicin could inhibit the expression quantity of FN1,ITGA6 and LAMB3 in normal and re-cultivated MDA-MB-231 cells. Conclusion： The tumor cells can be captured effectively by the microfluidic chip and re-cultured. The expressions of genes of the cells were not interfered remarkably for pre or post-captured. All of these would lay the foundation for the following related research such as biochemistry and molecular biology research,the efficacy evaluation analysis of antitumor drugs and so on.