人椎间盘细胞成骨潜能的研究

发布时间：2018-04-23 07:49

  本文选题：纤维环细胞 + 髓核细胞　； 参考：《中国人民解放军军医进修学院》2008年硕士论文

【摘要】： 研究背景: 特发性脊柱侧凸(adolescent idiopathic scoliosis,AIS)患者最终大多需要手术矫正,不仅治疗方案复杂,费用高,而且术中使用的各种融合材料包括自体骨、异体骨、人工骨等都存在着各种不足。椎间盘作为脊柱重要的连接单位,如果能通过人工干预使之骨化,则有望达到脊柱融合的目的。 椎间盘由纤维环和髓核两部分构成,椎间盘纤维环和髓核的共同特点是细胞外基质丰富而细胞数量较少,类软骨细胞存在于髓核及纤维环内层,成纤维细胞主要存在于纤维环的外层。当具有相应遗传物质基础的细胞处于有利于分化逆转的环境中,会出现转分化(transdifferentiation)。由一种分化状态变为另一种分化状态。 研究目的: 观察在实验室模拟的体外环境中椎间盘细胞的细胞学特性与表型表达的特点,观察在给予rh-BMP_2和bFGF进行人工干预后,体外培养的椎间盘细胞的表型表达和分化特性。 实验方法: 大体标本取自16-25岁青年患者,椎间盘本身无病变,标本离体后迅速送到实验室,仔细分离出纤维环和髓核,利用体外细胞培养技术建立腰椎间盘纤维环细胞和髓核细胞培养体系。观察这两种细胞在细胞因子rh-BMP_2和bFGF诱导下的成骨表型及生物学特性的变化。实验中按培养条件分为四个培养组:A组:DMEM/FBS培养液;B组:DMEM/FBS培养液+rh-BMP_2;C组:DMEM/FBS培养液+bFGF;D组:DMEM/FBS培养液+rh-BMP_2+bFGF。实验观察内容包括:细胞形态、生长曲线、碱性磷酸酶活力、Ⅰ型胶原表达、Ⅱ型胶原表达、骨钙素表达、茜素红染色等。 实验结果: 随着培养时间的延长,纤维环细胞呈明显的趋化性生长,极性排列,局部有细胞基质堆积。rh-BMP_2对纤维环细胞增殖无明显影响;能够提高碱性磷酸酶的活力;促进Ⅰ型胶原的表达;提高骨钙素的表达;促进钙盐的沉积,形成钙结节。bFGF能够明显促进细胞增殖;降低了碱性磷酸酶的活力;促进Ⅰ型胶原的表达;促进钙盐沉积,形成钙结节。当rh-BMP_2和bFGF联合使用时,能够促进细胞增殖;对碱性磷酸酶活力无明显影响;能够强力促进骨钙素、Ⅰ型胶原的表达和钙盐沉积,其效果优于单独使用任何一种细胞因子。 原代髓核细胞呈多角形,传代后变成星形或长梭形,培养后期呈明显的趋化性生长,极性排列。rh-BMP_2能够抑制细胞的增殖;提高骨钙素的表达;促进钙盐沉积,形成钙结节。bFGF能够促进细胞增殖;促进Ⅰ型胶原的表达;促进钙盐沉积,形成钙结节。当rh-BMP_2和bFGF联合使用时,能够促进细胞增殖;促进骨钙素、Ⅰ型胶原的表达和钙盐沉积,戍骨诱导效果最佳。四个培养组的Ⅱ型胶原表达均为阴性。 结论: rh-BMP_2对纤维环细胞体外有明确的成骨诱导作用,bFGF能够增强rh-BMP_2的成骨诱导作用。 rh-BMP_2对髓核细胞体外有明确的成骨诱导作用,bFGF能够增强rh-BMP_2的成骨诱导作用。
[Abstract]:Background: Most of the patients with idiopathic scoliosis who need surgical correction ultimately need surgical correction. Not only are the treatment schemes complex and costly, but also the various fusion materials used during the operation include autogenous bone, allogeneic bone, artificial bone and so on. Intervertebral disc, as an important junction unit of spine, can achieve spinal fusion if it can be ossified by artificial intervention. The intervertebral disc is composed of fibrous ring and nucleus pulposus. The common characteristic of disc fiber ring and nucleus pulposus is that the extracellular matrix is abundant and the number of cells is small. The chondroid cells exist in the nucleus pulposus and the inner layer of the fibrous ring. Fibroblasts mainly exist in the outer layer of the fibrous ring. When the cells with the corresponding genetic material base are in a favorable environment for differentiation reversal, transdifferentiation and transdifferentiation may occur. From one state of differentiation to another state of differentiation. Objectives of the study: The cytological and phenotypic characteristics of intervertebral disc cells were observed in laboratory simulated environment, and the phenotypic expression and differentiation characteristics of intervertebral disc cells cultured in vitro were observed after the artificial intervention of rh-BMP_2 and bFGF. Experimental methods: The specimens were taken from young patients aged 16-25 years, and there was no lesion in the intervertebral disc itself. The specimens were sent to the laboratory in vitro and carefully separated from the fibrous annulus and nucleus pulposus. The cell culture system of fibroannular cells and nucleus pulposus cells of lumbar intervertebral disc was established by cell culture in vitro. The changes of osteogenic phenotype and biological characteristics of these two kinds of cells induced by cytokines rh-BMP_2 and bFGF were observed. In the experiment, we divided into four groups according to the culture conditions: 1: a group: DMEM / FBS culture medium group B: DMEM / FBS medium rh-BMP2C group: DMEM / FBS medium bFGFGF group rh-BMP_2 bFGF. Cell morphology, growth curve, alkaline phosphatase activity, type 鈪，

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