SRPX2通过巨噬细胞促进人脐静脉内皮细胞血管生成能力的作用

SRPX2 promotes HUVEC angiogenesis via macrophages

目的:探究含sushi重复蛋白X连锁2蛋白(sushi repeat-containing protein X-linked 2,SRPX2)是否可通过巨噬细胞对血管生成产生影响及其可能的机制。方法:用shRNA-SRPX2及shRNA空载体转染HCT116细胞,并收集以上两种细胞和空白HCT116的细胞培养基作条件培养基,用3种条件培养基作用于人源单核细胞(THP-1)诱导的巨噬细胞,而后巨噬细胞与人脐静脉内皮细胞(HUVEC)共培养,并以Transwell迁移实验检测HUVEC迁移能力,以Matrigel基质胶管腔形成实验检测管腔形成能力。用Western blot检测巨噬细胞中局部黏着斑激酶(FAK)的表达水平。结果:在Transwell迁移实验中,HCT116敲降SRPX2基因的条件培养基与巨噬细胞作用,并与HUVEC共培养后,HUVEC迁移细胞数明显减少(P <0. 01)。而在Matrigel管腔形成实验中,HCT116敲降组形成的管腔的交叉点数、成网数、节点数也有所减少(P <0. 05)。FAK蛋白检测发现,SRPX2重组蛋白作用后的巨噬细胞总FAK表达量和FAK蛋白磷酸化水平有所增高。结论:SRPX2可能通过FAK相关通路影响巨噬细胞,进而间接影响血管内皮细胞的血管生成能力。

AIM: To investigate whether sushi repeat-containing protein X-linked 2 (SRPX2) can influence angiogenesis via macrophages, and its potential mechanism. METHODS: After transferring shRNA-SRPX2 and vacant shRNA vector into HCT116 cells, culture medium supernatant was extracted from three types of cells (HCT116-KD, HCT116-NC and HCT116-BC) as the conditioned medium. After culturing THP-1 induced macrophages in the conditioned medium, the macrophages were co-cultured with HUVECs for Transwell migration assay and Matrigel tube formation assay to verify the migration ability and tube formation ability of HUVECs. In addition, focal adhesion kinase (FAK) and phosphorylated FAK expression level in macrophages were measured by Western blot after activated by SRPX2 recombinant protein for 4 h. RESULTS:In migration assay, the number of HUVECs migrating to the lower chamber was significantly diminished in HCT116-KD group(P<0.01). In tube formation assay, numbers of junctions, meshes and nodes also decreased in the HCT116-KD group(P<0.05). As for the FAK expression, the expression of FAK as well as the phosphorylated level of FAK in macrophages elevated after interacted with SRPX2 recombination protein.CONCLUSION: SRPX2 can affect angiogenesis via macrophages, which is probably depends on FAK related signal pathways.