流体应力对兔骨髓MSCs向血管内皮细胞表型分化的影响

发布时间：2018-02-21 20:33

本文关键词： 骨髓间充质干细胞装置流体应力血管内皮细胞生长因子诱导分化表型　出处：《第三军医大学》2006年硕士论文　论文类型：学位论文

【摘要】： 骨移植材料植入体内后,必须尽快建立充分的血供,为骨组织修复或改建提供充足的营养,这在完成较大骨缺损修复时,尤为重要。血管生成是一个集细胞、细胞因子、细胞外基质、生物力学刺激等多种因素的复杂过程。既往的研究多侧重于应用VEGF,bFGF等促血管生成因子,但存在剂量不易调控,甚至导致血管瘤发生等缺陷。因此,探讨给予外源性的血管内皮细胞来促进局部血管生成成为新的研究策略。获取自体的血管内皮细胞存在较大的创伤,且细胞获取有限,而近年来研究发现具有多向分化潜能的骨髓间充质干细胞在体内外相应的诱导条件下,有进一步分化为内皮细胞表型,进而参与局部的血管生成可能。为此,本课题初步探讨流体应力对体外培养的兔骨髓间充质细胞向血管内皮细胞表型分化的影响。从而筛选出适宜的力学条件,为利用自体骨髓MSCs促血管生成奠定理论基础。一、目的模拟体内血流动力状态,建立正压条件下流体应力装置,并用此装置单独或联合血管内皮细胞生长因子来刺激兔BM-MSCs向血管内皮细胞表型的分化,为进一步应用该细胞来促血管生成的治疗创造条件。二、方法 1、通过梯度离心分离兔骨髓单核细胞,再结合定期换液的体外培养方法,获取兔BM-MSCs。通过测定细胞的增殖能力和成骨、成脂肪分化来鉴定细胞。 2、将目前常规使用的流体应力装置(平行板流动腔)中流体的负压状态,改良为正压状态,通过比较改良前后的平板流动腔内的压强、流量、流动的波形,分析兔骨髓MSCs增殖能力和表型变化来判断该装置对细胞生物学特性的影响。以不同的流体应力作用于平行板流动腔内的兔MSCs,分别于3、6、12和24h后通过胎盘兰染色检测活细胞比例,免疫组织化学染色检测细胞CD31阳性表达率。将活细胞比例超过80%并CD31阳性率超过70%的实验组条件,作为适宜的流体应力刺激大小和作用时间。
[Abstract]:Bone graft after implantation in vivo, must establish adequate blood supply as soon as possible, to provide adequate nutrition for bone tissue repair or reconstruction, which is especially important in the completion of repair of large bone defects,. Angiogenesis is a set of cells, cytokines, extracellular matrix, the complex process of many factors such as past. Biomechanical stimulation the study focuses on the application of VEGF, bFGF of Pro angiogenic factors, but the dose is not easy to control, and even lead to the development of hemangioma and other defects. Therefore, study of vascular endothelial cells to promote angiogenesis for local new strategy.
To obtain autologous vascular endothelial cells have great trauma, and limited access to cells, but recent studies found that the multilineage differentiation potential of bone marrow mesenchymal stem cells induced by the in vivo conditions, further differentiate into endothelial cell phenotype, and then participate in the angiogenesis of local potential. Therefore, this paper discussed the fluid effect of stress on vascular endothelial cells to differentiation of cultured rabbit bone marrow mesenchymal stem cells. In order to find out the suitable mechanical condition for the use of autologous bone marrow MSCs, lay a theoretical foundation for promoting angiogenesis.
First, the purpose
To simulate the in vivo hemodynamic state, establish positive pressure under the condition of fluid stress device, and the device used alone or in combination with differentiation of vascular endothelial growth factor to stimulate the rabbit BM-MSCs to the phenotype of vascular endothelial cells, to create conditions for the further application of the treatment of cells to promote angiogenesis.
Two, method
1, the rabbit bone marrow mononuclear cells were isolated by gradient centrifugation, and then the rabbit BM-MSCs. was obtained by in vitro culture with regular fluid exchange. The cells were identified by measuring cell proliferation and osteogenic differentiation.
2, the routine use of fluid stress device (parallel plate flow chamber) negative pressure fluid, modified for positive pressure, the pressure is modified and the plate flow cavity flow, flow waveform, to determine the effect of the device on the biological characteristics of cells of rabbit bone marrow MSCs proliferation and phenotype change.
With different fluid stress in rabbit MSCs parallel plate flow chamber, respectively at 3,6,12 and 24h after trypan blue staining to detect the proportion of living cells, immunohistochemical staining of CD31 cells. The positive expression rate of viable cells more than the experimental group conditions CD31 positive rate of 80% and more than 70%, as a suitable fluid the size and duration of stimulation.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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