摘要
目的:构建非受体型蛋白酪氨酸激酶Syk的真核表达载体,转染人乳腺癌细胞,使其在乳腺癌细胞中稳定表达,为进一步探讨Syk抑制肿瘤细胞侵袭转移的具体机制奠定基础。方法:构建syk基因的真核表达载体pcDNA3.1D/V5-His-TOPO/syk,以脂质体介导法转染Syk表达缺失的MDA-MB-231细胞,经G418筛选,获得syk基因稳定表达的MDA-MB-231/syk细胞。通过流式细胞术和RT-PCR技术检测转染细胞MMP-9的表达变化,用体外迁移和侵袭力实验测定转染细胞的迁移和侵袭能力。结果:构建了syk基因的真核表达载体pcDNA3.1D/V5-His-TOPO/syk,并在MDA-MB-231细胞中获得稳定表达。与野生型MDA-MB-231细胞相比,MDA-MB-231/syk细胞表达MMP-9的水平明显降低(P<0.01),并且该细胞的迁移和侵袭能力也明显降低(P<0.01)。结论:候选抑癌基因syk通过脂质体可有效转染MDA-MB-231细胞,MDA-MB-231/syk细胞迁移和侵袭能力明显降低,这为进一步研究Syk在体内的抑瘤作用和乳腺癌基因治疗奠定了基础。
Objective： To construct the eukaryotic expressing vector for human Spleen tyrosine kinase（Syk） and transfect it into human breast cancer cells for stable expression of Syk for the purpose of studying the mechanisms of Syk to inhibit tumor cells＇ invasion and metastasis.Methods：Eukaryotic expressing vector pcDNA3.1D/VS-His-TOPO/syk was constructed and transfected into MDA-MB-231 cells by lipofectamine protocols.After G418 selection,the cells MDA-MB-231/syk expressing Syk stably were obtained. Expression of MMP-9 on the cells was detected using flow cytometry and RT-PCR. Migratory and invasive abilities of the cells were analyzed with migration and invasion assays. Results：The eukaryotic expressing vector pcDNA3.1D/VS-His-TOPO/syk was constructed and syk was expressed in MDA-MB-231 cells. Compared with wild MDA-MB-231 cells,lower expression of MMP-9 was found in the cells（ P 〈 0.01）, with decreased migratory and invasive abilities（ P 〈 0.01 ）. Condusion： Potential tumor suppressor syk can be transfected into MDA-MB-231 cells by liposome efficiently. The migratory and invasive abilities of MDA-MB-231/syk cells reduced obviously. It has laid a solid foundation for further study of Syk suppressive effect in vivo and for gene therapy in breast cancer.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2008年第8期698-702,共5页
Chinese Journal of Immunology
关键词
乳腺癌
SYK
真核表达载体
迁移
侵袭
Breast cancer
Syk
Eukaryotic expression vector
Migration
Invasion
