体外培养的人创面肉芽组织成纤维细胞生物学特性的初步研究

龙艳，王达利，魏在荣等.体外培养的人创面肉芽组织成纤维细胞生物学特性的初步研究[J].中华整形外科杂志.2014, 30(3):203-209

肉芽组织;成纤维细胞;间质十细胞

龙艳，王达利，魏在荣，郭常敏

贵州省人民医院整形烧伤显微外科 ；遵义医学院附属医院烧伤整形外科,遵义,563003

2014年

203-209

知网,万方

目的 探讨人创面肉芽组织成纤维细胞增殖活力、CD分子表型、部分因子及相关蛋白表达和体外诱导分化能力等生物学特性,为进一步研究成纤维细胞在创面愈合早期的作用奠定基础.方法 采用机械法和酶消化法相结合分离培养创面肉芽组织成纤维细胞,倒置相差显微镜下观察细胞形态及增殖情况;绘制原代及第3代细胞生长曲线.流式细胞仪检测原代及第3代细胞表面CD分子的表达.免疫细胞化学检测波形蛋白、角蛋白19、CD31和第Ⅷ因子相关抗原的表达.进行成脂、成骨及成软骨细胞诱导分化能力的鉴定.结果 原代细胞呈短梭形、多角形或不规则形;传代后细胞主要以长梭形为主,排列规则,呈平行状、放射状、漩涡状生长;细胞增殖能力良好,有明显的对数生长期.在体外培养第1～4天,原代及第3代细胞增殖能力差异无统计学意义(P＞0.05),从第5天开始第3代比原代细胞增殖能力强(P＜0.01).细胞高表达CD105、CD73、CD90及C D44间充质干细胞阳性表面标志,不表达CD34、CD45、CD19、CD11b及HLA-DR间充质干细胞阴性细胞表面标志.原代细胞表达间充质干细胞阳性表面标志物比例分别为:CD105 55.22％、CD7399.03％、CD90 54.75％、CD44 98.62％,表达间充质干细胞阴性表面标志物CD34、CD45、CD19、CD11b及HLA-DR为0.65％.第3代细胞分别是CD105 98.28％、CD73 99.83％、CD90 99.52％、CD44 99.56％,CD34、CD45、CD19、CD11b及HLA-DR为0.61％.第3代表达间究质干细胞阳性表面标志的细胞比例呈现增高趋势.波形蛋白、CD31阳性表达;不表达角蛋白19和第Ⅷ因子相关抗原.成脂、成骨及成软骨细胞诱导分化阳性.结论 体外培养的人创面肉芽组织成纤维细胞表现出间充质干细胞样细胞生物学特性;且表达内皮细胞部分生物学标志物,创面血管内皮细胞通过内皮向间质转化(EndMT)可能是人创面愈合早期肉芽组织成纤维细胞的来源之一. Objective To explore the biological characteristics of fibroblasts in adult granulation tissue in vitro,including cell viability,CD phenotypic,factor and protein expression,and differentiation,so as to facilitate further research of the role of fibroblasts in early wound healing.Methods Fibroblasts were isolated from human granulation tissue and cultured by mechanical and enzymatic digestion method.The cell morphology and proliferation were observed under inverted phase contrast microscope.The first and third passages of cells'growth curve were drawn respectively.The surface markers (CD105,CD73,CD90,CD44,CD34,CD45.CD19,CD11 b,HLA-DR) of the first and third passage fibroblasts were identified by Flow Cytometry.And the expression of V imentin,C K19,C D31 and Factor Ⅷ were detected by immunocytochemistry.Results Primary cultured fibroblasts were short spindle,polygonal and irregular in shape.The morphology of fibroblasts were uniform by repeatedly passage cultured in vitro and showed spindle-shaped.The proliferative capacity of the fibroblasts were not significantly different,with logarithmic growth phase.From 1 to 4 days,The primary and third passage cells' proliferation was no difference (P ＞ 0.05),After the five days,the proliferative ability of third generation was better than the primary passage (P ＜ 0.01).All fibroblasts highly expressed mesenchymal stem cells' surface markers CD105,CD73,CD90 and CD44,and didnt express hematopoietic stem cells' surface markers CD34,CD45,CD19,CD11b and HLA-DR.The expression of mesenchymal stem cells' surface markers in third generation of cells were increased significantly.Immunocytochemistry showed positive expression of Vimentin,CD31,and negative expression of CK19 and Factor Ⅷ.Conclusions Fibroblasts in human granulation tissue show the biology characteristics of Mesenchymal Stem Cells.Some biological markers of endothelial cells are expressed in fibroblasts in granulation tissue.The fibroblasts may play an important role during the process of endothelial to mesenchymal transformation in early wound healing.。