兔同种异体脱钙骨复合骨髓间充质干细胞关节腔内培养软骨与同腔软骨的性状对比研究

发布时间：2018-01-09 21:59

本文关键词：兔同种异体脱钙骨复合骨髓间充质干细胞关节腔内培养软骨与同腔软骨的性状对比研究　出处：《安徽医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的采用兔骨髓间充质干细胞(BMSC)复合同种异体脱钙骨(DBM)体外培养和关节腔内培养组织工程软骨与同腔软骨的性状对比研究。方法1.采用同源雄性新西兰幼兔(4～6周龄)全骨髓贴壁筛选法培养原代BMSC,第三代细胞融合至约80%时培养基中加入成软骨诱导液:含转化生长因子(Transforming Growth Factor,TGF)-β1 10ng/ml、胰岛素样生长因子(Insulin-like Growth Factor,IGF)-1 20ng/ml、维生素C 50ng/ml。2.根据Urist提供的方法制作DBM并做扫描电镜(SEM)观察。3.将诱导后的BMSC种植于30个DBM支架培养并分别在体外(A组)及关节腔内(B组)培养。于4、8、12周分别取材,同时取同腔软骨(C组)作为对照,行SEM等大体形态观察并制石蜡切片行苏木素-伊红染色(Hematoxylin-Eosin Staining,HE)、甲苯胺蓝(Toluidine Blue,TB)染色、Masson三色染色、Ⅱ型胶原免疫组化等组织学观察并进行比较。结果1.本实验方法分离和培养的BMSC形态特征明显,呈长梭形,原代培养10～12天细胞融合约90%,增殖传代的细胞6～7天可达约90%融合。2.BMSC经定向诱导后表现出软骨细胞的特性。3.制备得到的DBM为三维立体疏松多孔的海绵样结构。4.A、B、C三组培养12w后,取出标本行SEM等大体观察、组织学切片观察及Ⅱ型胶原免疫组化检测:A组:SEM:标本表面凹凸不平,内部尚有部分空隙;HE染色见软骨细胞少量增生,胞核蓝染;甲苯胺蓝染色见软骨细胞排列无序,少量周围基质包绕;Masson染色阳性区域小,细胞排列无序;Ⅱ型胶原免疫组化见软骨细胞胞浆及胞外基质少量黄色颗粒。B组:SEM:标本表面较光滑;HE染色见软骨细胞增生,胞核蓝染;甲苯胺蓝染色见软骨细胞成串排列,软骨陷窝形成,周围基质包绕;Masson染色阳性,软骨细胞多,基质蓝染,按一定应力方向排列;Ⅱ型胶原免疫组化见细胞外基质中出现较多棕黄色颗粒,Ⅱ型胶原染色阳性。结论兔BMSC与同种异体DBM制备的细胞-支架复合物可在体外及膝关节腔内培养出组织工程软骨,关节腔内培养的软骨比体外培养的软骨更接近正常软骨。
[Abstract]:Objective to compare the characteristics of rabbit bone marrow mesenchymal stem cells (BMSC) combined with allogenic decalcified bone (DBM) cultured in vitro and articular cavity culture tissue engineered cartilage and the same cavity cartilage.
Methods 1. male New Zealand rabbits by homology (4 ~ 6 weeks) the whole bone marrow adherence screening method of primary cultured BMSC cells of the third generation fusion to about 80% when added to the medium of chondrogenic media containing transforming growth factor (Transforming Growth, Factor, TGF) 10ng/ml beta 1 and insulin-like growth factor (Insulin-like Growth Factor -1 20ng/ml, IGF), vitamin C 50ng/ml.2. according to the methods provided by Urist DBM and scanning electron microscopy (SEM) observation of.3. after induction BMSC grown in 30 DBM scaffold and respectively in vitro (A group) and intra-articular (B group). Cultured in 4,8,12 weeks were drawn at the same time take the same cavity cartilage (group C) as control, SEM for gross observation and paraffin sections were stained with hematoxylin eosin staining (Hematoxylin-Eosin Staining, HE (Toluidine), Blue, TB) toluidine blue staining, Masson staining, type II collagen immunohistochemical and histological view Compare and compare.
The results of the 1. experiment methods to isolate and culture BMSC morphology obviously, fusiform, cultured for 10~12 days about 90% cell fusion, cell proliferation and passage of 6~7 days can reach about 90%.2.BMSC fusion after oriented induction showed cavernous structure of.4.A, prepared by the cartilage cell characteristics of.3. DBM for the three dimensional system the three-dimensional porous B, C three groups after 12W, remove the specimens SEM, gross observation, histological observation and detection of type II collagen group: A group: SEM: specimen surface is uneven, there are some internal voids; HE staining of cartilage cell proliferation of small amount, blue stained nuclei; toluidine blue staining showed chondrocytes a small amount of disorder arrangement, surrounded by the matrix; Masson staining area is small, cells arranged in disorder; type II collagen immunohistochemical cartilage cell cytoplasm and extracellular matrix of small yellow particles in.B group: SEM: specimen surface is smooth; HE staining The proliferation of cartilage cells, blue stained nuclei; toluidine blue staining showed that the cartilage cells arranged in clusters, cartilage lacuna formation, surrounded by the matrix; Masson staining, cartilage cells, matrix stained blue, arranged in a certain stress direction; type II collagen immunohistochemical see more brown granules in the extracellular matrix, collagen staining positive.
Conclusion rabbit BMSC and allogenic DBM preparation of cell scaffold composite can produce tissue engineered cartilage in vitro and knee joint cavity, articular cartilage culture than in vitro cartilage closer to normal cartilage.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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