人脐带Wharton胶支架负载脂肪干细胞构建新型组织工程软骨的实验研究
发布时间：2018-10-26 19:13
【摘要】：目的:探索人脐带Wharton胶支架负载脂肪干细胞构建新型组织工程软骨的可行性,并评价该组织工程软骨修复兔膝关节全层软骨缺损的效果。方法:⑴从兔颈背部皮下脂肪分离得到脂肪干细胞,培养至P3代后通过流式细胞学鉴定其表面标记物,明确其干细胞来源。⑵普通培养至P3代后更换为诱导液(DMEM中加入10%FBS,10ng/ml TGF-β1,25ng/ml b FGF,10-7M地塞米松)成软骨方向诱导两周,收集细胞并调整终浓度为1×107/ml,将细胞接种至Wharton胶支架上继续三维诱导一周构建组织工程软骨。FDA-PI染色及Hoechst33258染色评估细胞在支架上的活力及增殖情况,扫描电镜观察细胞在支架上分布及黏附情况,甲苯胺蓝、番红O、Ⅱ型胶原免疫组化染色观察细胞外基质分泌情况。⑶于兔膝关节软骨滑车处形成1个直径4mm,深2mm的环形全层软骨缺损,设置空白组、单纯支架材料组及细胞支架复合体组。术后24周取材,缺损修复区行大体观察、组织学染色、糖胺多糖、总胶原含量检测及生物力学测定分析评价缺损修复情况。结果:⑴流式细胞学鉴定显示CD73、CD90、CD105阳性,CD34、CD45、HLA-DR阴性,提示所取细胞为脂肪来源干细胞;⑵FDA-PI染色及Hoechst33258染色可见支架上粘附的细胞分布均匀,活细胞较多,死细胞较少,支架深层也有部分细胞粘附。扫描电镜观察可见诱导后大量细胞黏附于支架孔隙内,呈圆形或椭圆形。组织学染色阳性提示诱导后细胞分泌大量细胞外基质;⑶术后24周细胞支架复合体组缺损区大部分被修复,缺损部位被软骨组织填充,修复区表面光滑。组织学染色显示有典型的透明软骨样结构,GAG、总胶原定量测定提示可达正常值的90%以上,生物力学测定提示与正常软骨相似。单纯支架材料组虽然缺损区大部分被修复,但修复区多为纤维软骨,仅有少量透明软骨样结构。空白组未能修复关节软骨缺损,纤维组织部分填充缺损,表面覆盖薄层纤维组织。结论:脂肪干细胞作为软骨组织工程种子细胞具有可行性,人脐带Wharton胶支架可作为良好软骨组织工程支架材料,实验构建的组织工程软骨在远期能够有效的修复关节软骨缺损。
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【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08;R68
本文编号：2296691
[Abstract]:% E7%9B%AE%E7%9A%84%3A%E6%8E%A2%E7%B4%A2%E4%BA%E8%84%90%E5%B8%A6Wharton%E8%83%B6%E6%94%AF%E6%9E%B6%E8%B4%9F%E8 % BD%E8%84%82%E8%82%AA%E5%B9%B2%E7%BB%86%E8%83%9E%E6%9E%84%E5%BB%BA%E6%96%B0%E5%9E%8B%E7%BB%84%E7%BB%87 % E5%B7%A5%E7%A8%8B%E8%BD%AF%E9%AA%A8%E7%9A%84%E5%8F%AF%E8%A1%8C%E6%80%A7%2C%E5%B9%B6%E8%AF%84%E4%BB%B7%E8 % AF%A5%E7%BB%84%E7%BB%87%E5%B7%A5%E7%A8%8B%E8%BD%AF%E9%AA%A8%E4%BF%AE%E5%A4%8D%E5%85%94%E8%86%9D%E5%85%B3 % E88A8A82B1B _ 1, E88A8A82B _ 1, E88A8A _ 8B _ 1, E88A _ 8A _ 8B _ 8A _ 8A _ 8B _ 7A _ 7F _ 9F _ 7AAE _ 7e _ 7e _ 7e _ 7F _ _ _ 2 _ _ _ 2 _ _ _ B395A E2, B395A, E882, E888, A838, E838, E832, E882, and E882, including E8882, A882, E588, and E8888, including E588, including E585, A882, E882, E888, and E8882, including E8882, A882, E888, E882, E888, 86E7A7A682B0, including E8887B3P3%, including E884882, E582, E886, E882, and E8882, including E884B982, B582, E582, and E8886, E832, E882, and E582, including E8887, B575, BBE887, B3, P3,%, E4BBA908, E4BBA9809A, E4BBA8FE589B5ADA6FB586E83E5ADA88FE5809A, including E6FE787, E6FB589B5ADA883E5ADA8, including E6BF878E589, E5809A, E9E5809A, and E9E9A9A9A589, including E585%, right, right. 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【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08;R68
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