shWnt5a重组慢病毒的制备及其对黑素瘤细胞侵袭的抑制作用  

Preparation of the recombinant lentiviral expression vector targeting human Wnt5a gene and its inhibitory effect on melanoma cell invasion

作　　者：赵亚军[1] 苗叶[1] 陈新雨[2] 王丽亚[2] 路凡[2] 廖文俊[1] 

机构地区：[1]第四军医大学西京医院皮肤科,陕西西安710032 [2]第四军医大学生物化学与分子生物学教研室,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2014年第5期462-465,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81072235)

摘　　要：目的构建下调人Wnt5a基因表达的重组质粒并制备重组慢病毒,感染黑素瘤细胞后,观察其对该细胞侵袭的影响。方法构建下调人Wnt5a基因表达的重组质粒pLKO.1-shWnt5a,利用pLKO.1慢病毒包装系统和HEK293T细胞包装、制备携带shWnt5a的重组慢病毒颗粒,并感染WM793B黑素瘤细胞,建立稳定低表达Wnt5a的WM793BWnt5a-黑素瘤细胞株。采取细胞迁移与侵袭实验检测Wnt5a对黑素瘤细胞侵袭的影响。结果成功制备了携带shWnt5a的重组慢病毒,建立低表达Wnt5a的稳定转染细胞株WM793BWnt5a-,细胞迁移与侵袭实验证明抑制Wnt5a基因的表达可以显著抑制黑素瘤的侵袭能力。结论下调Wnt5a基因的表达对黑素瘤的侵袭能力有明显的抑制作用。Objective To construct a recombinant lentiviral vector expressing small-hairpin RNA （shRNA） targeting human WntSa gene and investigate its silencing effect on WM793B human melanoma invasion. Methods Based on the sequence of human WntSa gene in GenBank, Wnt5a siRNA was designed and synthesized. The single-stranded primers were annealed to double-stranded oligonucleotide sequences and subcloned into linear pLKO. 1 lentMral plasmid digested by enzyme to produce pLKO. 1-shWntSa lentMral vector. After being identified by PCR a.nd sequencing, plasmid pLKO. 1- sh WntSa was transfected into HEK293T cells to package lentiviral particles. Human malenoma WM793B cells were infected by the lentiviral particles. Expression of sh WntSa in WM793B cells was detected using Western blotting. Then the TranswellTM invasion assay was performed to assess its effect on melanoma cell invasion. Results Lentivirus expressing shWntSa was successfully constructed and WM793B^Wn5a- , a strain of melanoma cells with low expression of WntSa was also established. TranswellTM invasion assay revealed that cell migration was inhibited in WntSa-inhibited melanoma cells. Conclusion Down-regulated Wnt5a expression exerts a significant inhibitory effect on the invasion of melanoma cells.

关 键 词：黑素瘤细胞 慢病毒 RNA干涉 shWnt5a 细胞侵袭 

分 类 号：R392.12[医药卫生—免疫学] Q257[医药卫生—基础医学]