人脐带间充质干细胞对放射线照射后人肺成纤维细胞中经典WNT/β-catenin通路的影响

Effect of human umbilical cord mesenchymal stem cells on lung fibroblasts exposed to radiation through the canonical WNT/β-catenin signaling pathway

目的探讨人脐带间充质干细胞(HUMSCs)对放射线照射后的人肺成纤维细胞(HLFs)影响的机制。方法 HLFs分为照射组(A)、照射+共培养组(B)和正常对照组(C),A组和B组在5Gy X射线照射后,B组与HUMSCs通过Transwell系统共培养。照射12、24、36h后western blot检测HLFs中α-SMA、GSK-3β、p-GSK-3β和胞核内β-catenin蛋白表达,ELISA法检测细胞培养液中WISP-1、SFRP-1蛋白表达。Real time PCR检测正常和B组第36h HUMSCs中SFRP-1 mRNA表达。结果放射线照射后第24h、36h,HLFs中α-SMA、p-GSK-3β、p-GSK-3β/GSK-3β、胞核内β-catenin,以及培养液中WISP-1蛋白表达水平,A组较C组明显升高,而B组较A组明显下降,差异有统计学意义(P<0.01);B组培养液中SFRP-1蛋白水平较A组、C组各时间点均显著升高,差异有统计学意义(P<0.05);B组第36h的HUMSCs中SFRP-1 mRNA表达明显高于正常HUMSCs,差异有统计学意义(P<0.01)。结论 HUMSCs可抑制放射线导致的HLFs中经典WNT/β-catenin通路的活化,并有可能通过该途径影响肺成纤维细胞的分化。

Objective To investigate the mechanism of human umbilical cord mesenchymal stem cells （HUMSCs） on irradiated human lung fibroblasts （HLFs） exposed to radiation through the canonical WNT/β-catenin signaling pathway. Methods HLFs were divided into the irradiated group （ A）, the irradiated ＋ cocuhured group （B） and the control group （C）. The groups A and B were irradiated by 5Gy X-rays, and then the group B was co- cultured with HUMSCs by Transwell system. 12, 24 and 36 hours after irradiation, the expression of ot-SMA, GSK- 313, p-GSK-313 and 13-catenin in nucleus protein were examined by Western blot respectively. The levels of SFRP-1 and WISP-1 protein in conditioned medium （CM） were examined by ELISA. The mRNA level of co-cultured HUM- SCs 36 hours after irradiation was examined by real time PCR. Results The protein levels of ct-SMA and some com- ponents of the canonical Wnt/-catenin signaling pathway, p-GSK3~/GSK313,13-catenin, and WISP-1 increased more significantly in the group A than in the group C, and they decreased more obviously in the group B than in the group C （ P 〈 O. 01 ）. ELISA showed an increase of SFRP-1 in CM of the group B in contrast with CM of the groups A and C （ P 〈0. 05）. The mRNA level of SFRP-1 increased in co-cultured HUMSCs 36 hours after irradiation while compared with normal HUMSCs （P 〈 O. O1 ）. Conclusion HUMSCs can prevent fibroblasts of postradiation from differentiating into myofibroblast, which may be through reversing the activation of canonical Wnt/-catenin signaling pathway.