中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2014年
2期
165-170
,共6页
组织构建%组织工程%脂肪干细胞%生物反应器%聚羟基乙酸%动态力学刺激培养%静态培养%国家自然科学基金
組織構建%組織工程%脂肪榦細胞%生物反應器%聚羥基乙痠%動態力學刺激培養%靜態培養%國傢自然科學基金
조직구건%조직공정%지방간세포%생물반응기%취간기을산%동태역학자격배양%정태배양%국가자연과학기금
stem cells%tissue engineering%bioreactor,urinary tract%flow cytometry
背景：理想的组织工程尿道替代物应具有良好的力学特性，足以承受长时间的尿液排泄冲击，而静态培养的尿路肌性管腔强度不佳。已有研究表明，力学刺激能够促进细胞生长和细胞外基质的分泌。目的：探讨生物反应器内构建组织工程化尿路肌性管腔的可行性。方法：酶消化法获取脂肪干细胞，经体外培养和扩增后，流式细胞技术检测细胞表面抗原，将脂肪干细胞接种于聚羟基乙酸上，形成细胞-材料复合物，体外培养1周后，将其置于生物反应器内培养，实验组予以动态力学刺激培养；对照组为静态培养，先采用基础培养基培养3周，而后用成肌诱导培养液诱导4周，行大体观察及组织学检测。结果与结论：流式细胞仪检测细胞表面CD90，CD44，CD105表达率分别为99.42%，98.12%，93.27%；CD34，CD45表达率分别为4.92%和0.38%，实验组培养的肌性管腔色泽明亮，管腔圆润，免疫组化染色显示，细胞材料复合物在诱导4周后，细胞表达结蛋白和α-平滑肌肌动蛋白阳性，细胞材料复合物胶原成分多。对照组构建的肌性管腔色泽暗淡，管腔轻度塌陷，细胞材料复合物胶原成分较少。提示脂肪干细胞复合聚羟基乙酸材料在生物反应器内动态培养可构建具有良好结构的尿路肌性管腔。
揹景：理想的組織工程尿道替代物應具有良好的力學特性，足以承受長時間的尿液排洩遲擊，而靜態培養的尿路肌性管腔彊度不佳。已有研究錶明，力學刺激能夠促進細胞生長和細胞外基質的分泌。目的：探討生物反應器內構建組織工程化尿路肌性管腔的可行性。方法：酶消化法穫取脂肪榦細胞，經體外培養和擴增後，流式細胞技術檢測細胞錶麵抗原，將脂肪榦細胞接種于聚羥基乙痠上，形成細胞-材料複閤物，體外培養1週後，將其置于生物反應器內培養，實驗組予以動態力學刺激培養；對照組為靜態培養，先採用基礎培養基培養3週，而後用成肌誘導培養液誘導4週，行大體觀察及組織學檢測。結果與結論：流式細胞儀檢測細胞錶麵CD90，CD44，CD105錶達率分彆為99.42%，98.12%，93.27%；CD34，CD45錶達率分彆為4.92%和0.38%，實驗組培養的肌性管腔色澤明亮，管腔圓潤，免疫組化染色顯示，細胞材料複閤物在誘導4週後，細胞錶達結蛋白和α-平滑肌肌動蛋白暘性，細胞材料複閤物膠原成分多。對照組構建的肌性管腔色澤暗淡，管腔輕度塌陷，細胞材料複閤物膠原成分較少。提示脂肪榦細胞複閤聚羥基乙痠材料在生物反應器內動態培養可構建具有良好結構的尿路肌性管腔。
배경：이상적조직공정뇨도체대물응구유량호적역학특성，족이승수장시간적뇨액배설충격，이정태배양적뇨로기성관강강도불가。이유연구표명，역학자격능구촉진세포생장화세포외기질적분비。목적：탐토생물반응기내구건조직공정화뇨로기성관강적가행성。방법：매소화법획취지방간세포，경체외배양화확증후，류식세포기술검측세포표면항원，장지방간세포접충우취간기을산상，형성세포-재료복합물，체외배양1주후，장기치우생물반응기내배양，실험조여이동태역학자격배양；대조조위정태배양，선채용기출배양기배양3주，이후용성기유도배양액유도4주，행대체관찰급조직학검측。결과여결론：류식세포의검측세포표면CD90，CD44，CD105표체솔분별위99.42%，98.12%，93.27%；CD34，CD45표체솔분별위4.92%화0.38%，실험조배양적기성관강색택명량，관강원윤，면역조화염색현시，세포재료복합물재유도4주후，세포표체결단백화α-평활기기동단백양성，세포재료복합물효원성분다。대조조구건적기성관강색택암담，관강경도탑함，세포재료복합물효원성분교소。제시지방간세포복합취간기을산재료재생물반응기내동태배양가구건구유량호결구적뇨로기성관강。
BACKGROUND:Ideal tissue-engineered urinary tract should have good mechanical properties to bear long-term attack of urine and excretion. Muscular conduit of urinary tract in static culture exerts poor strength. It is reported that mechanical stimuli promote cellular growth and secretion of extracellular matrix. OBJECTIVE:To investigate the feasibility of constructing tissue-engineered muscular conduit of urinary tract in bioreactor. METHODS:Adipose-derived stem cells were harvested by col agen enzyme method. After series culture and expansion in vitro, flow cytometric analysis was carried out to detect the immunophenotypes of adipose-derived stem cells. Then the cel-polyglycolic acid complex was constructed by seeding adipose-derived stem cells on polyglycolic acid fibers. After 1 week of in vitro culture, cel-polyglycolic acid complex was cultured in a bioreactor. The experimental group was subjected to pulsatile stimuli, while the control group was cultured in static state. After 3 weeks of in vitro culture in basic medium, the cel-polyglycolic acid complex was induced in the induced culture medium for 4 weeks, and then engineered tissue was examined both grossly and histological y. RESULTS AND CONCLUSION:Flow cytometry demonstrated that the adipose-derived stem cells expressed CD90 (99.42%), CD44 (98.12%) and CD105 (93.27%), but not CD34 (4.92%) or CD45 (0.38%). In the experimental group, tissue-engineered muscular conduit of urinary tract appeared bright color with a round lumen. Immunohistochemical staining showed that after cel-polyglycolic acid complex was induced for 4 weeks, the cells expressed desmin and α-smooth muscle actin. More col agen was found in the complex. In contrast, the control group appeared pale surface and its lumen col apsed slightly. Less col agen was in the complex. Tissue-engineered muscular conduit of urinary tract with good structure can be constructed in a bioreactor.
