CAJ | 학술논문

目的通过观察缺氧状态对瘢痕疙瘩成纤维细胞条件培养基诱导血管形成能力的影响,探讨缺氧微环境在瘢痕疙瘩浸润性生长中的作用.方法采用组织块法体外培养瘢痕疙瘩成纤维细胞,将其置入缺氧培养箱(2％O2)进行培养,并以常氧培养箱(20％ O2)培养作为对照,48 h后收集上清,并与内皮细胞培养基按1∶1混合后作为条件培养基.按常规方法培养人脐静脉血管内皮细胞.采用real time PCR和ELISA法,检测缺氧刺激对瘢痕疙瘩成纤维细胞促血管生成因子如血管内皮生长因子(vascular endothelial growth factor,VEGF)、血管生成素-1(angiopoietin-1,Ang-1)和periostin表达和分泌的影响;条件培养基孵育血管内皮细胞,采用CCK-8法测定其增殖情况,Transwell小室法检测其迁移和侵袭能力,基质胶小管形成实验观察其小管形成能力.结果缺氧刺激使瘢痕疙瘩成纤维细胞VEGF、Ang-1和periostin因子在mRNA水平分别增加了75％、43％、118％(P＜0.05),在分泌蛋白水平分别增加了30.2％、14.2％、19.5％ (P ＜0.05);缺氧状态下在瘢痕疙瘩成纤维细胞条件培养基孵育血管内皮细胞增殖的吸光度值,在1、2和3d分别为0.67±0.07、0.84±0.09和1.08±0.10,均高于其对照组(0.52 ±0.08、0.72±0.10和0.91±0.14,P＜0.05);缺氧状态下瘢痕疙瘩成纤维细胞条件培养基诱导血管内皮细胞的迁移、侵袭和小管形成数目分别为(73.2±8.9)、(56.3±12.5)、(9.66±1.96)个/高倍视野,均高于其对照组[(59.0±8.0)、(35.5±8.5)、(6.5±1.87)个/高倍视野,P＜0.05].结论　缺氧刺激增加瘢痕疙瘩成纤维细胞的促血管形成因子的合成分泌,其条件培养基显著增加血管内皮细胞的血管形成能力,提示缺氧微环境可能通过促新生血管形成在瘢痕疙瘩浸润性生长中起着重要作用.
목적 통과관찰결양상태대반흔흘탑성섬유세포조건배양기유도혈관형성능력적영향,탐토결양미배경재반흔흘탑침윤성생장중적작용.방법 채용조직괴법체외배양반흔흘탑성섬유세포,장기치입결양배양상(2％O2)진행배양,병이상양배양상(20％ O2)배양작위대조,48 h후수집상청,병여내피세포배양기안1∶1혼합후작위조건배양기.안상규방법배양인제정맥혈관내피세포.채용real time PCR화ELISA법,검측결양자격대반흔흘탑성섬유세포촉혈관생성인자여혈관내피생장인자(vascular endothelial growth factor,VEGF)、혈관생성소-1(angiopoietin-1,Ang-1)화periostin표체화분비적영향;조건배양기부육혈관내피세포,채용CCK-8법측정기증식정황,Transwell소실법검측기천이화침습능력,기질효소관형성실험관찰기소관형성능력.결과 결양자격사반흔흘탑성섬유세포VEGF、Ang-1화periostin인자재mRNA수평분별증가료75％、43％、118％(P＜0.05),재분비단백수평분별증가료30.2％、14.2％、19.5％ (P ＜0.05);결양상태하재반흔흘탑성섬유세포조건배양기부육혈관내피세포증식적흡광도치,재1、2화3d분별위0.67±0.07、0.84±0.09화1.08±0.10,균고우기대조조(0.52 ±0.08、0.72±0.10화0.91±0.14,P＜0.05);결양상태하반흔흘탑성섬유세포조건배양기유도혈관내피세포적천이、침습화소관형성수목분별위(73.2±8.9)、(56.3±12.5)、(9.66±1.96)개/고배시야,균고우기대조조[(59.0±8.0)、(35.5±8.5)、(6.5±1.87)개/고배시야,P＜0.05].결론　결양자격증가반흔흘탑성섬유세포적촉혈관형성인자적합성분비,기조건배양기현저증가혈관내피세포적혈관형성능력,제시결양미배경가능통과촉신생혈관형성재반흔흘탑침윤성생장중기착중요작용.
Objective To observe the effects of conditioned medium from keloid fibroblasts under hypoxia on angiogenesis,and to investigate the role of hypoxic microenvironment in invasive growth of keloid.Methods Primary keloid fibroblasts and human umbilical endothelial cells (HUVEC) were cultured as conventional method.Keloid fibroblasts were cultured either in a hypoxic incubator (2％ O2)for 48 h or in a normoxic incubator(20％ O2)as control.Then those cell culture mediums were collected and mixed with endothelial cell medium by the proportion of 1∶1 as conditioned medium.The mRNA and secreted protein of pro-angiogenic factors such as vascular endothelial growth factor (VEGF),angiopoietin1 (Ang-1) and periostin of keloid fibroblasts under hypoxia were detected by real time PCR and ELISA.The proliferation,migration and invasion,tube formation of HUVEC cultured with conditioned medium were evaluated by CCK-8 assay,Transwell assay and matrigel tube formation assay,respectively.Results Hypoxia increased the expression of VEGF,Ang-1 and periostin in both mRNA (increased by 75％,43％and 118％ respectively,P ＜ 0.05) and secreted protein (increased by 30.2％,14.2％ and 19.5％respectively,P ＜ 0.05) levels; the proliferations of HUVEC in hypoxic conditioned medium in 1,2 and 3 d were 0.67 ± 0.07,0.84 ± 0.09 and 1.08 ± 0.10 respectively,which were higher compared to those in control group (0.52 ±0.08,0.72 ±0.10 and 0.91 ±0.14,P ＜0.05) ; the numbers of migration,invasion and tube formation of HUVEC were (73.2 ± 8.9),(56.3 ± 12.5),(9.66 ± 1.96) cells/HP,which were higher compared to those in control group [(59.0 ± 8.0),(35.5 ± 8.5),(6.5 ± 1.87) cells/HP,P ＜0.05].Conclusions Hypoxia increases the expression of pro-angiogenic factors of keloid fibroblasts,and its conditioned medium under hypoxia could promote angiogenesis.The results suggest hypoxic microenvironment may play a significant role in the invasive growth of keloid by inducing angiogenesis.