发散式冲击波促进骨髓间充质干细胞增殖特性的实验研究

发布时间：2018-06-25 02:08

本文选题：体外发散式冲击波 + BMSC　；参考：《中国人民解放军医学院》2015年硕士论文

【摘要】：目的：通过应用低能量发散式冲击波(rESW)对兔骨髓间充质干细胞(BMSC)进行直接刺激,观察发散式冲击波对BMSC增殖特性的影响。本研究从BMSC冲击波刺激的反应出发,探求发散式冲击波促进关节软骨损伤修复的理论基础和作用机制。方法：在细胞实验部分,我们采用差速离心法和悬浮贴壁法从2～3月龄的新西兰白兔的胫骨骨髓中培养原代培养至P4～P6代。分别从形态学、表面BMSC,分子和分化能力(成脂、成骨)等方面对进行鉴定。用不同剂量的发散式BMSC冲击波对悬浮的进行直接刺激,根据冲击波剂量对实验进行分组,分为0BMSC四组(Bar为冲击波压力单位)。对冲击波刺激组与对照组的bar/1 bar/2 bar/3 bar分别进行CCK-8检测和BMSC检测比较其增殖活性,用统计学软件对实验CFU-F结果进行统计学分析。在动物实验部分,我们使用与之前实验等月龄的新西兰白兔制作膝关节软骨缺损模型,将每只兔子的左侧膝关节设为实验组,右侧膝关节设为对照组。实验组采用微骨折加2bar冲击波进行干预,而对照组只采用微骨折术。手术后24小时将骨髓等量冲出进行体外实验,在不同时间点对比CFU-F增殖聚集情况。在对数据结果进行分析统计时,我们使用BMSC软件并使用SPSS16.0软件对图像进行处理分析。ImageJ结果：通过骨髓分离技术我们可以获得原代在倒置相差显微镜下观BMSC,察细胞形态,类似成纤维细胞样聚集分布。在表面分子分析中,缺乏典型BMSC的造血细胞抗原CD45分子、CD14分子,也不表达CD79α和CD90,高表达CD45和CD81。在诱导分化实验中,可以被成功诱导为骨细胞、脂肪细胞,通过BMSC相应的染色得到证实,并检测相关转录因子表达。经不同的冲击波进行刺激后,表现出不同的增殖特性。无论是CCK-8检测还是BMSC检测,冲击波刺激CFU-F组都明显优于对照组,并且2Bar时其效应最强,其差异具有统计学意义(P0.05)。在动物体内实验进行CFU-F检测中,对术后24小时分离的MSc培养,并于第8天、11天、15天进行CFU-F检测,结果显示实验组增殖活性均优于对照组,其结果具有统计学意义(P0.05)。结论：1.我们发现兔BMSC在表面分子检测中CD90为阴性,这是与以往文献中的报道是不同的。2.发散式冲击波体外刺激BMSC具有促进其增殖和聚集作用,在冲击波剂量为2bar时其促增值作用最强。3.发散式冲击波对BMSC的作用呈现剂量相关性,其对于冲击波的软骨修复剂量具有指导作用。4.在动物实验中,采用微骨折术配合冲击波进行软骨缺损修复研究,为提出一种改良的微骨折术提供理论依据。
[Abstract]:Aim: to investigate the effect of low energy divergent shock wave (rESW) on the proliferation of rabbit bone marrow mesenchymal stem cells (BMSC). Based on the response of BMSC to shock wave stimulation, this study explored the theoretical basis and mechanism of divergent shock wave promoting articular cartilage repair. Methods: in the cell experiment, we used differential centrifugation and suspension adherent method from the tibia bone marrow of New Zealand white rabbits of 2 to 3 months old to primary culture to P4 and P6. Morphology, surface BMSCs, molecular and differentiation ability (adipogenic, osteogenic) were identified. The suspension was directly stimulated by different doses of divergent BMSC shock wave. According to the shock wave dose, the experiment was divided into four groups: 0BMSC (Bar was shock wave pressure unit). The proliferative activity of bar/1 bar/2 bar/3 bar in shock wave stimulation group and control group were detected by CCK-8 and BMSC respectively. The results of CFU-F were statistically analyzed by statistical software. In the part of animal experiment, we used New Zealand white rabbits with the same age to make cartilage defect model of knee joint. The left knee joint of each rabbit was set as experimental group and the right knee joint as control group. The experimental group was treated with microfracture plus 2bar shock wave, while the control group was treated with microfracture. Bone marrow was washed out 24 hours after operation in vitro to compare the proliferation and aggregation of CFU-F at different time points. In the analysis and statistics of the data results, we use BMSC software and SPSS 16.0 software to analyze the image processing. ImageJ results: through bone marrow separation technology, we can obtain primary BMSCs under inverted phase contrast microscope, observe cell morphology. Similar to fibroblast-like aggregation distribution. In surface molecular analysis, CD45, CD14, CD79 伪 and CD90, and CD45 and CD81were not expressed in typical BMSC. In the induction and differentiation experiment, osteocytes and adipocytes could be successfully induced, confirmed by the corresponding staining of BMSC, and the expression of related transcription factors was detected. After different shock waves were stimulated, they showed different proliferative characteristics. Whether CCK-8 test or BMSC test, shock wave stimulation CFU-F group was significantly better than the control group, and the effect of 2Bar was the strongest, the difference was statistically significant (P0.05). In vivo CFU-F test, the MSC isolated 24 hours after operation was cultured and CFU-F was detected on the 8th day, 11th day and 15th day. The results showed that the proliferative activity of the experimental group was better than that of the control group, and the results were statistically significant (P0.05). Conclusion 1. We found that the CD90 of rabbit BMSC was negative in surface molecular detection, which was different from previous reports. The proliferation and aggregation of BMSC were stimulated by divergent shock wave in vitro. When the dose of shock wave was 2bar, the effect of proliferation was the strongest. 3. The effect of divergent shock wave on BMSC is dose-dependent, and it has a guiding effect on cartilage repair dose of shock wave. In animal experiments, microfracture combined with shock wave was used to repair cartilage defects, which provided a theoretical basis for improving microfracture.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R687.4

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