兔自体外周血间充质干细胞对腱—骨界面愈合影响的实验研究

发布时间：2017-12-31 02:43

  本文关键词：兔自体外周血间充质干细胞对腱—骨界面愈合影响的实验研究　出处：《安徽医科大学》2012年硕士论文　论文类型：学位论文

  更多相关文章： 外周血间充质干细胞 腱—骨界面愈合 前交叉韧带 交叉韧带重建

【摘要】：目的探讨兔自体外周血间充质干细胞（MSCs）对前交叉韧带（ACL）重建后腱-骨界面愈合的影响。 方法以粒细胞集落刺激因子作为外周血MSCs动员剂，体外分离、培养、扩增获得外周血MSCs。48只4~6月龄新西兰白兔按1-48编号，随机均分为A、B、C三组，以后肢右膝关节作为研究对象，采用趾长屈肌腱作为ACL重建移植物。A组兔将外周血间充质干细胞-生物蛋白胶（FG-MSCs）混合注入兔右膝ACL重建模型股骨道顶端间隙内；B组兔仅注射生物蛋白胶；C组兔交叉韧带重建后不做其他处理。分别在2周、4周、8周、12周各时间点每组各处死4只兔子进行解剖学观察，取材制成石蜡切片行苏木素-伊红染色(Hematoxylin-Eosin Staining,HE)、甲苯胺蓝染色(Toluidine Blue Staining,TB)、Masson三色染色（Masson trichrome staining）进行组织形态学观察，并进行腱-骨界面各时间点成纤维细胞计数及组织形态学Buark评分，以了解腱-骨界面愈合情况。 结果（1）①A组：术后2周可见腱-骨界面间隙由疏松结缔组织填充，可见少量成纤维细胞，移植物与骨道之间分离；术后4周可见大量疏松结缔组织、成纤维细胞和少量类软骨细胞及胶原纤维，新生骨形成，腱-骨间隙已不可见；术后8周在腱-骨界面可见大量类软骨细胞，出现Sharpey纤维集结成束，胶原纤维合成增多,排列较规则；术后12周腱-骨间形成致密结缔组织，可见大量排列有序的Sharpey纤维以及纤维软骨、钙化软骨形成，未出现潮线。②B、C组：术后2周腱-骨界面仅见少量疏松结缔组织，少量成纤维细胞增生,未见胶原纤维，同时腱-骨界面明显分离；术后4周腱-骨间可见少量疏松结缔组织及少量新生骨细胞长入，散在分布的类软骨细胞和胶原纤维，且未见与骨道垂直连接；术后8周腱-骨界面为较多类软骨细胞及胶原纤维，Sharpey纤维散在分布；术后12周有较多排列无序的类软骨细胞出现，，胶原纤维密集排列不规则，Sharpey纤维排列紊乱。（2）术后各时间点三组腱-骨界面高倍镜下（HE染色）成纤维细胞计数资料采用单向方差分析及SNK法检验：A组*与B组**和A组*与C组***在各时间点比较均有统计学意义（P＜0.05），而B组**与C组***在各时间点比较无统计学意义。（3）基于高倍镜下腱-骨界面的纤维血管组织和Sharpey纤维评定的组织形态学Buark评分统计学资料采用Wilcoxon秩和检验：12周内A组与B组组织形态学Buark评分中位数间的差异有统计学意义（P＜0.01），即A组组织形态学Buark评分较B组高。 结论兔自体外周血MSCs通过顶端注射的方式能够促进腱-骨界面的愈合。
[Abstract]:Objective to investigate the effect of autologous peripheral blood mesenchymal stem cells (MSCs) on the healing of tendon bone interface after the reconstruction of anterior cruciate ligament (ACL) in rabbits.
Methods with granulocyte colony-stimulating factor for peripheral blood MSCs mobilization agent, in vitro isolation, culture, amplification of peripheral blood MSCs.48 4~6 month old New Zealand white rabbits by 1-48 number, were randomly divided into A, B, C three groups, after limb knee joint as the research object, as ACL reconstruction graft in group.A the peripheral blood mesenchymal stem cells and fibrin glue with flexor digitorum longus tendon (FG-MSCs) mixed into ACL model of rabbit femur reconstruction of right knee top clearance; B group were only injected with fibrin glue; no other treatment in group C after anterior cruciate ligament reconstruction. In 2 weeks, 4 weeks, 8 week, 12 weeks each time point in each group were sacrificed 4 rabbits were observed in paraffin section, hematoxylin eosin staining (Hematoxylin-Eosin, Staining, HE), toluidine blue staining (Toluidine Blue Staining, TB), Masson trichrome staining (Masson trichrome staining) organization form In order to understand the healing of the tendon bone interface, the fibrous cell count and the histomorphology Buark score at the time point of the tendon bone interface were observed.
Results (1) A group: 2 weeks after surgery, the tendon bone interface by loose connective tissue filled the gap, a small amount of fibroblasts, separation between graft and bone; 4 weeks after operation showed a large amount of loose connective tissue, fibroblasts and a small amount of cartilage cells and collagen fibers, new bone formation that tendon bone gap is not visible; 8 weeks after operation in the tendon bone interface showed a large number of chondrocytes, Sharpey fiber bundles of collagen synthesis increased, a regular arrangement; 12 weeks after operation, the tendon bone formation of dense connective tissue, showing a large number of ordered Sharpey fiber and fiber cartilage. Calcified cartilage formation, without tidal line. The B, C group: after 2 weeks of tendon bone interface but only a small amount of loose connective tissue, a small amount of fibroblast proliferation, collagen fibers were at the same time, the tendon bone interface were separated; after 4 weeks of tendon bone can be seen between the small amount of loose connective tissue And a small amount of new bone cells, scattered cartilage cells and collagen fibers, and no bone and vertical connection; 8 weeks after operation, the tendon bone interface into chondrocytes and collagen fibers, Sharpey fibers were distributed; 12 weeks after surgery more cartilage cell disorder arrangement, dense collagen fibers arranged in irregular, disordered arrangement of Sharpey fiber. (2) at each time point after operation three groups of tendon bone interface at high magnification (HE staining) fibroblast count data using one-way analysis of variance and SNK test: A group and B group and A * * * * * * * in the group and C group at each time point were statistically significant (P < 0.05), B group and C group * * * * * in each time point is not significant. (3) fibrovascular tissue and Sharpey fibers evaluated at high magnification, the tendon bone interface morphology Buark Score statistical data using the Wilcoxon rank test based on: 12 weeks There was significant difference in the median of Buark score between group A and group B (P < 0.01), that is, the histomorphological Buark score of group A was higher than that of the B group.
Conclusion the autologous peripheral blood MSCs can promote the healing of the tendon bone interface by injection of the apical injection.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R681;R-332

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