中华耳鼻咽喉头颈外科杂志
中華耳鼻嚥喉頭頸外科雜誌
중화이비인후두경외과잡지
CHINESE JOURNAL OF OTORHINOLARYNGOLOGY HEAD AND NECK SURGERY
2014年
6期
473-478
,共6页
杨润琴%明磊国%邓志宏%金岩
楊潤琴%明磊國%鄧誌宏%金巖
양윤금%명뢰국%산지굉%금암
喉黏膜%间充质干细胞%声带%成肌细胞%细胞分化
喉黏膜%間充質榦細胞%聲帶%成肌細胞%細胞分化
후점막%간충질간세포%성대%성기세포%세포분화
Larnyngeal mucosa%Mesenchymal stem cell%Vocal cords%Myoblasts%Cell differentiation
目的 将喉黏膜间充质干细胞(laryngeal mucosal mesenchymal stem cells,LM-MSC)向成肌细胞诱导分化,为声带部分或全部缺失的组织工程修复寻求优势种子细胞.方法 从正常会厌舌面黏膜分离培养获得LM-MSC,流式细胞仪检测细胞表面分子标志CD44、CD105、CD90、CD29、CD34和CD45;向成脂、成骨细胞分化鉴定细胞多向分化潜能,油红O、茜素红染色鉴定成脂、成骨分化情况;加入成肌诱导液向成肌细胞方向分化,于成肌诱导分化后1周和3周时行免疫荧光化学染色,诱导后1周、3周和6周行实时定量RT-PCR分析,以分别定性和定量分析成肌分化相关蛋白及基因Myod1、Myogenin、肌球蛋白重链(myosin heavy chain,MyHc)的表达.结果 本实验分离培养获得的LM-MSC可贴壁生长,呈长梭形;高表达间充质干细胞表面分子标志物:CD44(100.0％),CD105(90.4％),CD90(99.9％),CD29 (93.0％),低表达造血系干细胞表面分子标志物:CD34(0.4％)和CD45(1.3％);LM-MSC在体外定向向成脂、成骨分化14 d和21 d后有大量串珠样脂滴和矿化结节生成;成肌细胞初始标志蛋白Myod1和特异性标志蛋白Myogenin,MyHc分别于成肌诱导分化后1周和3周出现,RT-PCR结果与免疫荧光化学染色结果一致,并且相关基因持续表达于成肌诱导分化后6周.结论 LM-MSC符合间充质干细胞的特性,并可定向分化为成肌细胞,有潜能成为声带组织工程修复的优势种子细胞.
目的 將喉黏膜間充質榦細胞(laryngeal mucosal mesenchymal stem cells,LM-MSC)嚮成肌細胞誘導分化,為聲帶部分或全部缺失的組織工程脩複尋求優勢種子細胞.方法 從正常會厭舌麵黏膜分離培養穫得LM-MSC,流式細胞儀檢測細胞錶麵分子標誌CD44、CD105、CD90、CD29、CD34和CD45;嚮成脂、成骨細胞分化鑒定細胞多嚮分化潛能,油紅O、茜素紅染色鑒定成脂、成骨分化情況;加入成肌誘導液嚮成肌細胞方嚮分化,于成肌誘導分化後1週和3週時行免疫熒光化學染色,誘導後1週、3週和6週行實時定量RT-PCR分析,以分彆定性和定量分析成肌分化相關蛋白及基因Myod1、Myogenin、肌毬蛋白重鏈(myosin heavy chain,MyHc)的錶達.結果 本實驗分離培養穫得的LM-MSC可貼壁生長,呈長梭形;高錶達間充質榦細胞錶麵分子標誌物:CD44(100.0％),CD105(90.4％),CD90(99.9％),CD29 (93.0％),低錶達造血繫榦細胞錶麵分子標誌物:CD34(0.4％)和CD45(1.3％);LM-MSC在體外定嚮嚮成脂、成骨分化14 d和21 d後有大量串珠樣脂滴和礦化結節生成;成肌細胞初始標誌蛋白Myod1和特異性標誌蛋白Myogenin,MyHc分彆于成肌誘導分化後1週和3週齣現,RT-PCR結果與免疫熒光化學染色結果一緻,併且相關基因持續錶達于成肌誘導分化後6週.結論 LM-MSC符閤間充質榦細胞的特性,併可定嚮分化為成肌細胞,有潛能成為聲帶組織工程脩複的優勢種子細胞.
목적 장후점막간충질간세포(laryngeal mucosal mesenchymal stem cells,LM-MSC)향성기세포유도분화,위성대부분혹전부결실적조직공정수복심구우세충자세포.방법 종정상회염설면점막분리배양획득LM-MSC,류식세포의검측세포표면분자표지CD44、CD105、CD90、CD29、CD34화CD45;향성지、성골세포분화감정세포다향분화잠능,유홍O、천소홍염색감정성지、성골분화정황;가입성기유도액향성기세포방향분화,우성기유도분화후1주화3주시행면역형광화학염색,유도후1주、3주화6주행실시정량RT-PCR분석,이분별정성화정량분석성기분화상관단백급기인Myod1、Myogenin、기구단백중련(myosin heavy chain,MyHc)적표체.결과 본실험분리배양획득적LM-MSC가첩벽생장,정장사형;고표체간충질간세포표면분자표지물:CD44(100.0％),CD105(90.4％),CD90(99.9％),CD29 (93.0％),저표체조혈계간세포표면분자표지물:CD34(0.4％)화CD45(1.3％);LM-MSC재체외정향향성지、성골분화14 d화21 d후유대량천주양지적화광화결절생성;성기세포초시표지단백Myod1화특이성표지단백Myogenin,MyHc분별우성기유도분화후1주화3주출현,RT-PCR결과여면역형광화학염색결과일치,병차상관기인지속표체우성기유도분화후6주.결론 LM-MSC부합간충질간세포적특성,병가정향분화위성기세포,유잠능성위성대조직공정수복적우세충자세포.
Objective To investigate the myogenic differentiation of laryngeal mucosal mesenchymal stem cells (LM-MSCs) and the possibility of LM-MSCs as new alternative seed cells for laryngeal tissue engineering.Methods LM-MSCs were separated from normal epiglottis mucosa and the cell surface markers including CD44,CD105,CD90,CD29,CD34 and CD45 were analyzed through flow cytometry.The osteogenesis and adipogenesis differentiation of LM-MSCs were investigated by oil red staining and alizarin red S staining.Immunofluorescence staining and RT-PCR were used to detect the expressions of myogenic differentiation markers including Myod1,Myogenin and myosin heavy chain (MyHc).Results The separated LM-MSCs were in a fibrocyte-like form with long fusiform shape and grew adherently.The expression rates of cell surface markers LM-MSCs were CD44 (100.0％),CD105 (90.4％),CD90 (99.9％),CD29 (93.0％),CD34 (0.4％) and CD45 (1.3％) respectively.A number of beaded lipid drops and mineral deposition were observed after 14 days of adipogenesis differentiation and 21 days of osteogenesis differentiation.Myod1,Myogenin and MyHc genes appeared after 1 week and 3 weeks of myogenesis differentiation respectively.Conclusions The LM-MSCs have the properties of mesenchymal stem cells and could be differentiated into myoblasts,providing with the possibility to repair the damaged vocal cords with LM-MSCs through tissue engineering techniques.
