辐射诱导成纤维细胞FAP表达对放射治疗后瘢痕复发的影响

Effect of Radiation-induced FAP Expression on Fibroblasts Involved in Scar Recurrence after Radiotherapy

目的探讨成纤维细胞活化蛋白(FAP)的变化水平对电子线照射后瘢痕疙瘩复发的作用,并阐明其相关作用机制。方法将8例患者瘢痕疙瘩术后组织处理成边长为3 mm的正方体组织块,移植到NCG小鼠双侧背部,建立瘢痕疙瘩异种移植模型。照射组为移植后1周,对右侧移植物给予10 Gy电子线照射,左侧为未照射组,观察4周;通过分子生物学和免疫组织化学分析其电子线照射前后的波形蛋白(Vimentin)和FAP的表达水平,对比移植物照射后的血管变化。通过照射前后转录组测序结果,推测FAP对辐射后瘢痕疙瘩复发的作用机制,并分离原代成纤维细胞,进行体外试验加以验证。结果小鼠移植物免疫组织化学检测结果显示,与未照射组比较,照射组照射后4周的组织中FAP表达显著增加,CD31表达下降(P<0.01)。同时荧光定量PCR结果显示,对原代成纤维细胞进行照射后,Vimentin和FAP的mRNA水平明显上调(P<0.01)。CCK8法检测结果,与未照射组比较,照射组在450 nm处吸光度(A)值更高(P<0.05)。流式细胞术分析结果显示,与未照射组比较,照射组照射后4周,能明显促进成纤维细胞进入S期(P<0.05或P<0.01),加速成纤维细胞的细胞周期进程。结论辐照可诱导成纤维细胞FAP表达上调,同时加速其细胞周期进程,可能是放疗后瘢痕疙瘩复发的原因之一。

Objective To investigate the effect of fibroblast activation protein alpha(FAP)on keloid recurrence after electron irradiation,and to elucidate its possible mechanism.Method The keloid postoperative tissues of 8 patients were treated into 3 mm square cubes,and the keloid xenotransplantation model was established by bilateral back transplantation in NCG mice.One week after transplantation,the right graft was treated with 10 Gy electron beam irradiation,the left was the control group and lasted for 4 weeks observation.The mRNA and protein expression levels of Vimentin and FAP before and after radiation were analyzed by molecular biological methods and immunohistochemistry,and the vascular changes after radiation of the grafts were compared.The mechanism of FAP on keloid recurrence after irradiation was speculated by transcriptomic detection before and after irradiation,and verified by in vitro experiments with primary fibroblasts.Results Immunohistochemical results showed that FAP expression significantly increased in the tissues irradiated by electron line 4 weeks after irradiation comparing with the control group.On the contrary,the expression of CD31 decreased in the irradiated group 4 weeks after exposure.At the same time,the results of fluorescence quantitative PCR showed that the mRNA levels of Vimentin and FAP were significantly up-regulated after irradiation of primary fibroblasts(P<0.01).The CCK8 results showed that the absorbance of the irradiated group was higher at 450 nm than that of the unirradiated group(P<0.05).Flow cytometry analysis showed that irradiation significantly promoted to enter S phase after 4 weeks which accelerated the cell cycle of fibroblasts(P<0.05 or P<0.01).Conclusion The irradiation can induce the up-regulation of FAP expression in fibroblasts and accelerate the cell cycle,which may be one of the reasons for the recurrence of keloid after radiotherapy.