CAJ | 학술논문

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. 目的探討人創麵肉芽組織成纖維細胞增殖活力、CD分子錶型、部分因子及相關蛋白錶達和體外誘導分化能力等生物學特性,為進一步研究成纖維細胞在創麵愈閤早期的作用奠定基礎.方法採用機械法和酶消化法相結閤分離培養創麵肉芽組織成纖維細胞,倒置相差顯微鏡下觀察細胞形態及增殖情況;繪製原代及第3代細胞生長麯線.流式細胞儀檢測原代及第3代細胞錶麵CD分子的錶達.免疫細胞化學檢測波形蛋白、角蛋白19、CD31和第Ⅷ因子相關抗原的錶達.進行成脂、成骨及成軟骨細胞誘導分化能力的鑒定.結果原代細胞呈短梭形、多角形或不規則形;傳代後細胞主要以長梭形為主,排列規則,呈平行狀、放射狀、漩渦狀生長;細胞增殖能力良好,有明顯的對數生長期.在體外培養第1～4天,原代及第3代細胞增殖能力差異無統計學意義(P＞0.05),從第5天開始第3代比原代細胞增殖能力彊(P＜0.01).細胞高錶達CD105、CD73、CD90及C D44間充質榦細胞暘性錶麵標誌,不錶達CD34、CD45、CD19、CD11b及HLA-DR間充質榦細胞陰性細胞錶麵標誌.原代細胞錶達間充質榦細胞暘性錶麵標誌物比例分彆為:CD105 55.22％、CD7399.03％、CD90 54.75％、CD44 98.62％,錶達間充質榦細胞陰性錶麵標誌物CD34、CD45、CD19、CD11b及HLA-DR為0.65％.第3代細胞分彆是CD105 98.28％、CD73 99.83％、CD90 99.52％、CD44 99.56％,CD34、CD45、CD19、CD11b及HLA-DR為0.61％.第3代錶達間究質榦細胞暘性錶麵標誌的細胞比例呈現增高趨勢.波形蛋白、CD31暘性錶達;不錶達角蛋白19和第Ⅷ因子相關抗原.成脂、成骨及成軟骨細胞誘導分化暘性.結論體外培養的人創麵肉芽組織成纖維細胞錶現齣間充質榦細胞樣細胞生物學特性;且錶達內皮細胞部分生物學標誌物,創麵血管內皮細胞通過內皮嚮間質轉化(EndMT)可能是人創麵愈閤早期肉芽組織成纖維細胞的來源之一.
목적 탐토인창면육아조직성섬유세포증식활력、CD분자표형、부분인자급상관단백표체화체외유도분화능력등생물학특성,위진일보연구성섬유세포재창면유합조기적작용전정기출.방법 채용궤계법화매소화법상결합분리배양창면육아조직성섬유세포,도치상차현미경하관찰세포형태급증식정황;회제원대급제3대세포생장곡선.류식세포의검측원대급제3대세포표면CD분자적표체.면역세포화학검측파형단백、각단백19、CD31화제Ⅷ인자상관항원적표체.진행성지、성골급성연골세포유도분화능력적감정.결과 원대세포정단사형、다각형혹불규칙형;전대후세포주요이장사형위주,배렬규칙,정평행상、방사상、선와상생장;세포증식능력량호,유명현적대수생장기.재체외배양제1～4천,원대급제3대세포증식능력차이무통계학의의(P＞0.05),종제5천개시제3대비원대세포증식능력강(P＜0.01).세포고표체CD105、CD73、CD90급C D44간충질간세포양성표면표지,불표체CD34、CD45、CD19、CD11b급HLA-DR간충질간세포음성세포표면표지.원대세포표체간충질간세포양성표면표지물비례분별위:CD105 55.22％、CD7399.03％、CD90 54.75％、CD44 98.62％,표체간충질간세포음성표면표지물CD34、CD45、CD19、CD11b급HLA-DR위0.65％.제3대세포분별시CD105 98.28％、CD73 99.83％、CD90 99.52％、CD44 99.56％,CD34、CD45、CD19、CD11b급HLA-DR위0.61％.제3대표체간구질간세포양성표면표지적세포비례정현증고추세.파형단백、CD31양성표체;불표체각단백19화제Ⅷ인자상관항원.성지、성골급성연골세포유도분화양성.결론 체외배양적인창면육아조직성섬유세포표현출간충질간세포양세포생물학특성;차표체내피세포부분생물학표지물,창면혈관내피세포통과내피향간질전화(EndMT)가능시인창면유합조기육아조직성섬유세포적래원지일.
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.