大鼠体外培养脂肪干细胞对鼓膜成纤维细胞的影响  

Effects of in vitro cultured adipose-derived stem cells of rats on the tympanic membrane fibroblasts

作　　者：雷霏[1] 孙建军[2] 刘阳[2] 姜丹泉[2] 

机构地区：[1]吉林大学中日联谊医院耳鼻咽喉头颈外科,长春130031 [2]海军总医院耳鼻咽喉头颈外科

出　　处：《中华耳鼻咽喉头颈外科杂志》2010年第7期587-591,共5页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：基金项目：中国博士后科学基金（096-4317）

摘　　要：目的 观察大鼠脂肪干细胞（adipose-derived stem cell,ASC）对鼓膜成纤维细胞增殖、迁移的作用,初步探讨脂肪干细胞用于鼓膜损伤修复治疗的可行性及意义.方法 以Wistar大鼠为研究对象,分别分离、培养及鉴定ASC和鼓膜成纤维细胞.通过transwell共培养方法检测ASC对鼓膜成纤维细胞增殖的影响;通过transwell细胞迁移实验检测ASC对鼓膜成纤维细胞迁移速度的影响.激光共聚焦显微镜下评估各项检测指标.结果 大鼠ASC表面标志免疫荧光鉴定结果显示CD44、CD29表达阳性,CD34表达阴性;鼓膜成纤维细胞表面标志鉴定结果显示波形蛋白（vimentin）表达阳性.细胞增殖测量结果显示,与ASC共培养的鼓膜成纤维细胞增殖速度比对照组明显加快,细胞计数差异具有统计学意义（t=6.75,P=0.003）.细胞迁移测量结果显示,在ASC条件培养基作用下,鼓膜成纤维细胞于1、2.5和4 h在Matrigel胶内位置距底部聚碳酸酯膜孔的平均距离比对照组均明显缩小,即迁移速度明显加快,差异具有统计学意义（P值均〈0.05）;5 h穿越至聚碳酸酯膜下表面的细胞数明显多于对照组,组间差异具有统计学意义（P〈0.05）.结论 ASC可能通过旁分泌作用促进鼓膜成纤维细胞增殖和迁移,将有利于鼓膜纤维层的修复.Objective To explore the effects of rats adipose-derived stem cell （ASC） on the proliferation and migration of fibroblasts from the tympanic membrane, and to discuss the possibility and significance of therapy with ASC for tympanic membrane （TM） healing and regeneration. Methods Wistar rats were sacrificed, and then the isolation, culture and identification of both ASC and the TM fibroblasts were performed respectively. To verify the effect of ASC on fibroblasts proliferation, transwell coculture system was used. To examine the effect of ASC on fibroblasts migration, cell migration assay with transwell was also applied. All the data were analyzed under a confocal laser scan microscopy system.Results Immunofluorescence of cell surface markers indicated that rats ASC were positive for both of CD44 and CD29, but negative for CD34. The rat TM fibroblasts were positive for vimentin. The fibroblasts cocultured with ASC proliferated faster than the fibroblasts of control group, and the difference of the cell counting number between the two groups was significant （ t = 6. 75, P = 0. 003 ）. Compared with the control group, the fibroblasts cultured with ASC conditioned culture medium migrated significantly faster, and the space between the fibroblasts and the polycarbonate membrane pore was significantly shortened at different time point （ 1, 2. 5 and 4 h, P 〈 0. 05 ）. The cell number of the fibroblasts that had migrated through the polycarbonate membrane had been significantly increased 5 h after migration （ P 〈 0. 05 ）. Conclusion ASC might promote TM fibroblasts proliferation and migration by paracrine activation, and it will faciliate the regeneration of TM fibrous layer.

关 键 词：间质干细胞 成纤维细胞 鼓膜 细胞 培养的 大鼠 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]