兔骨髓间充质干细胞与Buffy coat体外软骨分化的比较分析

发布时间：2018-04-22 18:53

本文选题：间充质干细胞 + Buffy　；参考：《延边大学》2017年硕士论文

【摘要】：目的:比较分析兔骨髓间充质干细胞(Mesenchymal Stem Cells,MSC)与含有单核细胞、生长因子和细胞因子等的Buffy coat在体外软骨分化,为Buffy coat治疗软骨损伤提供实验依据。方法:动物选取新西兰系大白兔12只(16周龄,体重3±0.5kg),随机分为:MSC组(阴性对照组,n=3),采集骨髓的间充质干细胞(MSC),不添加转化生长因子-β(Transforing growth factor-β,TGF-β)的诱导下培养。Buffy coat组(实验组,n=6),采用Buffy coat不经过体外的传代培养,在软骨诱导液下培养。MSC+TGF组(阳性对照组,n=3),采集骨髓的间充质干细胞,添加TGF-β的诱导下培养。通过大体观察、组织学观察、糖胺多糖(Glycosaminoglycan,GAG)检测,和体外软骨BERN的评分比较三组在体外分化的能力。结果:1)大体观察:MSC组培养的软骨部分已经溶解,边缘模糊;MSC+TGF组与Buffy coat组基本没有溶解现象,边缘圆润、清晰。2)番红O染色镜下观察:MSC组软骨细胞生长缓慢,分布不均匀,数量极少,有极少量GAG的分泌现象出现,分泌不规则;有少数细胞可以找到软骨细胞特有的Cartilage lacuna结构。Buffy coat组与MSC+TGF组软骨细胞生长速度较快,细胞分布较均匀,细胞数量较多,表面较平整、周围较圆润,有较多GAG分泌现象出现,基本分布在软骨细胞周围;有较多细胞可以找到Cartilage lacuna结构。3)糖胺多糖(GAG)含量测定:MSC 组:7.78± 1.79μg,Buffy coat 组:22.83±3.11μg,MSC+TGF 组:24.05±3.35μg。三组间存有显著差异,MSC 组与 MSC+TGF 组、MSC 与 Buffy coat 组,组间存在显著差异(P0.05);Buffy coat组与MSC+TGF组,组间无显著差异(P0.05)。4)体外软骨 BERN 评分:MSC 组:2.83± 1.16,Buffy coat 组:6.16±1.16,MSC+TGF组:6.68±0.81。三组间存在显著差异,MSC组与MSC+TGF组、MSC与Buffy coat组,组间存在显著差异(P0.05),Buffy coat组与MSC+TGF组,组间无显著差异(P0.05)。结论:Buffy coat具有良好的体外软骨分化能力,可以解决目前体外分化软骨必须添加生长因子这个难题,为Buffy coat在临床应用提供了实验依据。
[Abstract]:Aim: to compare and analyze the cartilage differentiation of rabbit bone marrow mesenchymal Stem cells (MSCs) and Buffy coat containing monocytes, growth factors and cytokines in vitro, so as to provide experimental evidence for the treatment of cartilage injury by Buffy coat. Methods: twelve New Zealand white rabbits aged 16 weeks were selected. Body weight was 3 卤0.5 kg 路kg ~ (-1), and was randomly divided into two groups: 1 / growth group (negative control group). MSCs were collected from bone marrow and cultured without TGF- 尾-尾 -Transforing growth factor- 尾 (TGF- 尾). Buffy coat group (experimental group) was cultured without Buffy coat in vitro. Mesenchymal stem cells (MSCs) were collected from bone marrow and cultured with TGF- 尾. The ability of differentiation of the three groups in vitro was compared by gross observation, histological observation, glycosaminoglycan glycosaminoglycan gag test and cartilage BERN score in vitro. Results (1) the chondrocytes cultured in the TGF group were dissolved, but there was no dissolution in the TGF group and the Buffy coat group. The edges were smooth and clear. Under the microscope, the chondrocytes in the Buffy coat group grew slowly and distributed unevenly. A few cells could find the specific Cartilage lacuna structure of chondrocytes. Buffy coat group and MSC TGF group had a faster growth rate, more evenly distributed cells and more cells. The surface was smooth, the surrounding was smooth, and there were more GAG secretion phenomena, which were mainly distributed around the chondrocytes, and the Cartilage lacuna structure. 3) glycosaminoglycan (Gagg) content could be found in the Cartilage lacuna group. The content of Cartilage was 7. 78 卤1. 79 渭 g / m Buffy coat group and 20. 83 卤3. 11 渭 g / m GAG TGF group. There was significant difference among the three groups. There was significant difference between the two groups (P0.05 Buffy coat group and MSC TGF group). There was no significant difference among the three groups (P0.05. 4) the in vitro cartilage BERN score was 2.83 卤1.16 Buffy coat group, 6.16 卤1.16 MSC coat group, 6.68 卤0.81MSC TGF group. There was significant difference among the three groups. There was no significant difference between the two groups (P0.05Buffy coat group and MSC TGF group). Conclusion coat has a good ability of cartilage differentiation in vitro, which can solve the problem that growth factor must be added to the differentiation cartilage in vitro, and provide experimental basis for the clinical application of Buffy coat.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R687;R318.08

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