成纤维细胞与明胶海绵支架三维共培养的实验研究

发布时间：2018-09-05 20:10

【摘要】：目的：应用组织块法体外培养原代成纤维细胞,观察细胞形态、生长特点、增殖能力以及细胞与医用可吸收明胶海绵体外共培养情况,为进一步研究成纤维细胞在组织工程学的应用打下基础。 方法：取4-6月龄新西兰大白兔,取其皮下筋膜组织,组织块法培养原代成纤维细胞。传代至第3代。倒置显微镜观察细胞形态,免疫组织学染色后共聚焦显微镜观察细胞形态,分别应用MTT和CCK8试剂盒研究细胞生长增殖规律,并对两种方法进行比较,Ⅰ/Ⅲ型胶原蛋白染色观察其分泌蛋白情况,PKH26标记下用扫描电镜观察成纤维细胞与明胶海绵体外共培养情况。 结果：用筋膜组织行组织块法培养可成功得到大量原代成纤维细胞,此法取材广泛,过程简单,成纤维细胞于体外培养时增殖活跃。分别用CCK8与MTT法连续观察7天,于细胞周期的第4-5天后细胞出现增殖平台期(平均4.1±2.0天和4.6±1.7天)。两种方法比较无明显统计学差异(P0.05)。免疫组织学染色后共聚焦显微镜观察可见细胞为典型的成纤维细胞形态：大胞体,多突起的纺锤形或星形的扁平细胞,细胞核呈规则的卵圆形,多核仁。Ⅰ/Ⅲ型胶原蛋白染色阳性。PKH26荧光染料可很好标记成纤维细胞细胞壁,染色率100%。扫描电镜观察成纤维细胞在明胶海绵支架中生长旺盛,增殖活跃。 结论：1.皮下筋膜组织块法培养成纤维细胞是一种简单易行的方法,可于体外获得大量成纤维细胞2.明胶海绵是一种良好的组织工程学韧带支架,与成纤维细胞贴敷率高,体外共培养良好。
[Abstract]:Objective: to culture primary fibroblasts in vitro by tissue mass method, and observe the morphology, growth characteristics, proliferation ability and co-culture of cells with medical absorbable gelatin in vitro. It will lay a foundation for further research on the application of fibroblasts in tissue engineering. Methods: primary fibroblasts were cultured from 4-6 months old New Zealand white rabbits with subcutaneous fascia tissue. Pass on to the third generation. The morphology of cells was observed by inverted microscope and confocal microscope after immunohistochemical staining. The law of cell growth and proliferation was studied by MTT and CCK8 kit, and the two methods were compared. Type 鈪，

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