稳定表达鼠源PLEKHA1细胞系的建立及其对细胞迁移的影响

目的：构建稳定表达鼠源PLEKHA1的RAW264.7小鼠巨噬细胞系，探讨PLEKHA1过表达对巨噬细胞迁移的影响。方法：根据小鼠PLEKHA1基因序列设计引物，克隆其编码区序列，酶切后插入pCDH载体，在293FT细胞中进行病毒的包装，用获得的高滴度慢病毒感染RAW264.7细胞，建立能稳定高效表达PLEKHA1的RAW264.7细胞系；在此基础上，观察PLEKHA1对巨噬细胞迁移的影响。结果：经基因克隆、酶切、连接后，构建了鼠源PLEKHA1重组慢病毒表达载体，包装病毒感染RAW264.7细胞，经嘌呤霉素筛选后免疫印迹检测，RAW264.7细胞中PLEKHA1的蛋白表达提高近30倍；同时，发现PLEKHA1的过表达影响RAW264.7细胞的迁移。结论：构建了稳定表达小鼠PLEKHA1的RAW264.7细胞系，PLEKHA1过表达降低RAW264.7细胞的迁移能力。

Establishment of Cell Line Stably Expressing Murine PLEKHA1 and Their Migration Function

Objective： To establish the murine RAW264.7 macrophages stablely expressing murine PLEKHA1, then analyze migration in this cell line. Methods： Primers were designed according to the murine PLEKHA1 gene, then the ORF of murine PLEKHA1 gene were amplified and cloned into pCDH vector. The vector was trans-fected into 293FT cells with lentiviral packaging plasmids. Then the infectious lentiviral was obtained, which in-fected the RAW264.7 cells. Following selection the stable PLEKHA1 over expression cells and control cells. Re-sults： The vector of pCDH-PLEKHA1 was constructed by inserting the ORF of murine PLEKHA1 gene into pCDH vector. Stable PLEKHA1 over expression cell line was established and the expression of PLEKHA1 was 30 folds higher than control cells. Transwell migration assay demonstrated that migrating ability was depressed in the cell line. Conclusion： RAW264.7 cell lines stably expressing murine PLEKHA1 were constructed, and overexpres-sion of PLEKHA1 can inhibite the migration of RAW264.7 cell.