骨膜蛋白在眼睑基底细胞癌相关成纤维细胞与眼睑正常成纤维细胞中的表达差异

Expression differences and significance of periostin in eyelid basal cell carcinoma associated fibroblasts andnormal fibroblasts

目的比较对已分离、培养、纯化、鉴定的眼睑基底细胞癌相关成纤维细胞(basal cell carcinoma associated fibroblasts,BCAFs)与眼睑正常成纤维细胞(normal fibroblasts,NFs)中骨膜蛋白(periostein,PN)表达差异。方法取生长良好的第3代纯化的BCAFs和NFs,用胰蛋白酶调制细胞悬液的浓度均为20×103个·m L^(-1),按每孔2 m L细胞悬液接种六孔板。以无血清培养基培养48 h后收集上清液,通过酶联免疫吸附试验(ELISA)检测BCAFs与NFs上清液中PN的含量;以盖玻片置于六孔板底制作细胞爬片,应用免疫荧光细胞化学染色技术检测BCAFs与NFs中PN的表达。结果眼睑BCAFs与NFs上清液中PN的含量分别为(9.26±2.35)μg·L^(-1)和(2.57±0.41)μg·L^(-1);细胞免疫荧光化学染色检测PN表达显示,眼睑BCAFs中强于NFs,差异有统计学意义(P<0.05)。结论眼睑BCAFs较NFs高表达分泌PN,提示PN在眼睑基底细胞癌的局部侵袭生长中起着一定作用。

Objective To investigate the expression differences and significance of periostin(PN)in eyelid basal cell carcinoma associated fibroblasts(BCAFs)andnormal fibroblasts(NFs)after separation,culture,purification and identification.Methods The third generation of purified BCAFs and NFs was selected,and the concentrations of cell suspensions were modulated to 20×10 6 L-1 by trypsin,and then the cell suspension were seeded and cultured in 6-well plate by 2 mL per well.The cell culture supernatants were collected when BCAFs and NFs were cultured by serum-free medium for 48 h,then the content of PN in cell culture supernatants from BCAFs and NFs was detected by enzyme-linked immunosorbent assay(ELISA).The glass coverslips were placed at the bottom of the 6-well plate to make cell slides,and then the expression of PN in BCAFs and NFs cells were tested by immunofluorescence staining.Results ELISA showed that the content of PN in cell culture supernatants from BCAFs and NFs was(9.26±2.35)μg·L-1 and(2.57±0.41)μg·L-1.And the expression level of PN in BCAFs tested by immunofluorescence staining technology was higher than that in NFs cells,and the differences were statistically significant(all P<0.05).Conclusion The expression and secretion of PN in the eyelid BCAFs were highly enhanced when compared with NFs,suggesting that periostein may promote or inhibit the occurrence and development of the eyelid basal cell carcinoma in the microenvironment of the eyelid basal cell carcinoma.