Cas9蛋白对亲骨转移人乳腺癌细胞株生物学性质和超微结构的影响

目的：构建表达Cas9蛋白的亲骨转移人乳腺癌细胞株，并研究Cas9蛋白的表达对细胞生物学性质和超微结构的影响，为利用CRISPR/Cas9技术研究乳腺癌骨转移分子机制提供实验基础。方法：通过慢病毒载体介导Cas9蛋白基因转染亲骨转移人乳腺癌细胞株MDA-MB-231BO，通过G418筛选转染细胞，采用流式细胞术、Western blot、免疫细胞化学验证Cas9基因转染是否成功；运用实时无标记动态细胞分析技术和细胞划痕实验检测Cas9蛋白表达对细胞增殖及迁移的影响；透射电镜观察Cas9蛋白表达对细胞超微结构的影响。结果：成功构建稳定表达Cas9蛋白的亲骨转移人乳腺癌细胞株MDA-MB-231BO-Cas9，Cas9蛋白的表达对细胞的增殖、迁移和超微结构无明显影响。结论：MDA-MB-231BO-Cas9细胞可用于CRISPR/Cas9技术进一步研究。

Effect of Cas9 Protein on Biological and Ultrastructural Characteristics of the Human Bone-seeking Breast Cancer Cell Line

Objective:To generate the human bone-seeking breast cancer cell line expressing Cas9 protein and study its biological and ultrastructural properties for the research of breast cancer cells' bone Metastasis using CRISPR-Cas9 technology. Methods:Cas9 protein gene was transfected into the human bone-seeking breast cancer cell line MDA-MB-231BO by lentiviral vector. After G418 screening, MDA-MB-231BO-Cas9 cell line which can constantly express Cas9 protein was obtained and examined by flow cytometry, Western blot and immunocytochemistry; Cell proliferation and migration were verified by real time cellular analysis and wound-healing assay; the effect of Cas9 expression on the ultrastructure of cells was observed under the transmission electron microscope. Results:The human bone-seeking breast cancer cell line MDA-MB-231BO-Cas9 expressing Cas9 protein has been constructed successfully, and the expression of Cas9 has no effect on cell's proliferation, migration and ultrastructure. Conclusion:MDA-MB-231BO-Cas9 cell line can be used for the further research using the CRISPR/Cas9 technology.