生长分化因子-5及碱性成纤维细胞生长因子诱导家兔椎间盘髓核细胞成骨的研究
发布时间：2018-07-20 13:09
【摘要】：背景：腰椎滑脱是骨科的常见病症之一，以反复腰痛为主要临床表现，严重影响患者的生活质量。而腰椎滑脱患者最终大多数需要手术矫正，术中用于脊柱融合的材料存在各种不足，如果能通过人工干预使椎间盘骨化，则有望达到脊柱融合的目的。有研究显示，GDF-5在软骨发生和长骨发育中具有重要的作用，能诱导多种细胞向成软骨和成骨细胞分化，，这表明其可诱导类软骨细胞-髓核细胞向成骨细胞分化。 目的：本实验通过建立家兔椎间盘髓核细胞的体外培养模型，观察髓核细胞的细胞学特性和生物学特性，然后在给予rhGDF-5和bFGF进行人工干预后，探讨rhGDF-5和bFGF对髓核细胞表型表达和分化特性的影响，并为直接诱导椎间盘髓核细胞成骨行椎间融合提供理论依据。 方法：1.从健康家兔取出椎间盘髓核，用Ⅱ型胶原酶消化法分离原代椎间盘髓核细胞后，单层培养并以胰酶消化法传代，采用差速贴壁法进行纯化。2.观察髓核细胞在细胞因子rhGDF-5和bFGF诱导下成骨表型表达和细胞学特性的变化。按试验中的细胞培养条件分为四个培养组：A组：DMEM/15%FBS培养液；B组：DMEM/15%FBS培养液+rhGDF-5；C组：DMEM/15%FBS培养液+bFGF；D组：DMEM/15%FBS培养液+rhGDF-5+bFGF。3.利用光学显微镜观察细胞形态，MTT法测定生长曲线，免疫组化观察Ⅰ型胶原和Ⅱ型胶原的表达，放射免疫技术测定骨钙素的表达，茜素红染色等。 结果：初贴壁的髓核细胞呈短梭形、多角形，融合后呈旋涡状，传代后呈长梭形或星形，培养后期呈明显的趋化性生长、极性排列。大约于第五代时细胞出现生长滞缓，即出现老化现象。rhGDF-5能够抑制细胞的增殖；增加骨钙素的表达；促进钙盐的沉积，形成钙结节。bFGF能够促进细胞的增殖；对骨钙素表达无明显影响；促进Ⅰ型胶原的表达；促进钙盐沉积，形成钙结节。当联合使用rhGDF-5和bFGF时，能够促进细胞增殖；能够促进Ⅰ型胶原、骨钙素的表达和钙盐的沉积，其效果要比单独使用任一细胞因子都好。四个组的Ⅱ型胶原表达均为阴性。 结论：1.本实验成功建立家兔髓核细胞体外培养模型。原代培养的髓核细胞主要呈类软骨细胞样表型,逐渐出现向成纤维细胞转分化。在细胞传至第五代时，出现老化现象。2. rhGDF-5能成功诱导体外髓核细胞成骨，bFGF能加强rhGDF-5对髓核细胞的成骨诱导作用。
[Abstract]:Background: lumbar spondylolisthesis is one of the common diseases in orthopedic department. However, most of the lumbar spondylolisthesis patients need surgical correction, and the materials used in spinal fusion have various shortcomings. If the intervertebral disc ossification can be made by artificial intervention, it is expected to achieve the purpose of spinal fusion. Some studies have shown that GDF-5 plays an important role in chondrogenesis and long bone development and can induce the differentiation of many kinds of cells into chondroblasts and osteoblasts, which indicates that GDF-5 can induce the differentiation of chondroblast-nucleus pulposus cells into osteoblasts. Objective: to observe the cytological and biological characteristics of nucleus pulposus cells in vitro by establishing a rabbit model of nucleus pulposus cells cultured in vitro, and then to observe the effects of rhGDF-5 and bFGF. To investigate the effects of rhGDF-5 and bFGF on phenotypic expression and differentiation of nucleus pulposus cells, and to provide theoretical basis for direct induction of intervertebral fusion of nucleus pulposus cells. Method 1: 1. The nucleus pulposus of the intervertebral disc was removed from the healthy rabbits. Primary nucleus pulposus cells were isolated by type 鈪
本文编号：2133625
[Abstract]:Background: lumbar spondylolisthesis is one of the common diseases in orthopedic department. However, most of the lumbar spondylolisthesis patients need surgical correction, and the materials used in spinal fusion have various shortcomings. If the intervertebral disc ossification can be made by artificial intervention, it is expected to achieve the purpose of spinal fusion. Some studies have shown that GDF-5 plays an important role in chondrogenesis and long bone development and can induce the differentiation of many kinds of cells into chondroblasts and osteoblasts, which indicates that GDF-5 can induce the differentiation of chondroblast-nucleus pulposus cells into osteoblasts. Objective: to observe the cytological and biological characteristics of nucleus pulposus cells in vitro by establishing a rabbit model of nucleus pulposus cells cultured in vitro, and then to observe the effects of rhGDF-5 and bFGF. To investigate the effects of rhGDF-5 and bFGF on phenotypic expression and differentiation of nucleus pulposus cells, and to provide theoretical basis for direct induction of intervertebral fusion of nucleus pulposus cells. Method 1: 1. The nucleus pulposus of the intervertebral disc was removed from the healthy rabbits. Primary nucleus pulposus cells were isolated by type 鈪
本文编号：2133625
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