破骨细胞与成纤维细胞体外相互作用研究

发布时间：2018-07-26 14:05

【摘要】：破骨细胞是体内唯一可以执行骨吸收功能的细胞,来源于造血细胞系前体,单核-巨噬细胞。破骨细胞在骨代谢和重塑中起着关键的作用。破骨细胞介导的骨吸收与成骨细胞介导的骨形成,通过一系列复杂的机制形成偶联,构成了骨量的动态平衡。成纤维细胞具有向成骨分化的潜能,如今成纤维细胞的在骨化中的作用日益被人们重视。成纤维细胞(fibroblast,FB)是动物结缔组织中最常见的细胞,由胚胎时期的间充质细胞(mesenchymal cell)分化而来,主要功能是分泌细胞外基质和胶原,维持结缔组织结构网络。在组织创伤修复和新血管生成过程中发挥着关键作用。另外,成纤维细胞参与了许多纤维化疾病的发病过程,如肺纤维化,肾纤维化,和硬皮病。成纤维细胞还具有一定的成骨能力,参与骨折愈合,并在强直性脊柱炎等疾病的异位骨化过程中发挥重要作用。成纤维细胞是如何向成骨样细胞分化并引起软组织骨化的机制尚不明确。但在成骨细胞的分化过程中破骨细胞起着至关重要的作用。破骨细胞条件培养基可通过Wnt/BMP信号通路和趋化因子鞘氨醇-1-磷酸诱导间充质干细胞向成骨细胞分化。成纤维细胞与成骨细胞属于同一个谱系。破骨细胞可以诱导成骨细胞分化,因此我们推断成纤维细胞介导的异位骨化过程中,破骨细胞可能参与其中并发挥了重要作用。研究目的:本研究的目的是了解人外周血单核细胞来源的破骨样细胞与人韧带成纤维细胞通过非接触的方式进行的体外相互作用。本课题着重(1)研究破骨细胞对人韧带成纤维细胞矿化的诱导作用,并初步探索其作用机制;(2)观察人韧带成纤维细胞条件培养基是否可以诱导破骨细胞前体向破骨分化。内容和方法:用Ficoll-Paque密度梯度离心发分离外周血单个核细胞;并采用免疫磁珠法分选CD14~+单核细胞;CD14~+细胞经M-CSF和RANKL两种因子诱导向破骨样细胞分化;通过形态观察、耐酒石酸盐酸性磷酸酶(TRAP)染色和扫描电镜观察骨吸收,鉴定破骨细胞特性。同时用胶原酶消化法从人的脊柱韧带中原代培养成纤维细胞。破骨细胞条件培养基(Osteoclasts conditioned medium)处理韧带成纤维细胞,BCIP/NBT碱性磷酸酯酶显色试剂盒染色检测成纤维碱性磷酸酶(ALP)的表达,茜素红染色观察矿化结节,并评估矿化水平。以正常培养基培养的成纤维细胞作对照,采用mRNA表达谱芯片和microRNA芯片检测成纤维细胞mRNA和microRNA(miRNA)表达谱的变化,并进行生物信息学分析,如差异表达基因的GO分析和pathway分析。利用另一例患者韧带中原代培养的成纤维细胞,重复条件培养基处理和芯片实验。用成纤维细胞的条件培养基培养破骨细胞前体,即CD14~+单核细胞,观察破骨细胞的分化。TRAP染色试剂盒检测TRAP阳性表达率,RT-PCR检测破骨分化标志基因的表达情况。结果:经破骨样细胞条件培养基培养的成纤维细胞碱性磷酸酶表达阳性率较对照组显著增高,茜素红染色观察到明显的矿化结节。实验组对照组相比,mRNA和miRNA的表达均有显著差异。mRNA表达谱芯片检测结果,522个基因上调,415个基因下调两倍以上;miRNA芯片结果,28个miRNA上调,59个miRNA下调2倍以上。重复实验中mRNA芯片检测结果:3707个mRNA上调,3251个mRNA下调;microRNA芯片重复结果:23条miRNA上调,36条miRNA下调。成纤维细胞条件培养基处理后的CD14~+单核细胞,具有40%的TRAP阳性率,同时经RT-PCR检测到TRAP和其他破骨细胞分化标志基因的表达。结论:破骨样细胞可在体外诱导韧带成纤维细胞发生矿化,而且这一过程中有miRNA参与表达调控。同时成纤维细胞条件培养基可诱导CD14~+细胞向破骨细胞分化,成纤维细胞与破骨细胞在代谢和分化过程中存在相互作用。
[Abstract]:Osteoclasts are the only cells in the body that can perform bone absorption, derived from the precursors of the hematopoietic cell line, monocyte macrophage. Osteoclast plays a key role in bone metabolism and remodeling. Osteoclast mediated bone absorption and osteoblast mediated bone formation, formed by a complex mechanism to form a bone mass. Dynamic balance. Fibroblasts have the potential to differentiate into osteogenic differentiation. Nowadays, the role of fibroblasts in ossification is becoming increasingly important. Fibroblast (FB) is the most common cell in the connective tissue of animal, which is differentiated from the embryonic mesenchyme (mesenchymal cell), and the main function is to secrete the extracellular matrix. And collagen, maintaining a connective tissue structure network. It plays a key role in tissue trauma repair and new angiogenesis. In addition, fibroblasts are involved in the pathogenesis of many fibrotic diseases, such as pulmonary fibrosis, renal fibrosis, and scleroderma. Fibroblasts also have a certain osteogenic ability to participate in fracture healing and in ankylosis. There is an important role in the process of heterotopic ossification of diseases such as spondylitis. The mechanism of fibroblast is how to differentiate into osteoblast like cells and cause soft tissue ossification. However, osteoclasts play a vital role in the differentiation of osteoblasts. The condition of osteoclast conditioned culture can be mediated through Wnt/BMP signaling pathway and chemotaxis. Factor sphingosine -1- phosphoric acid induces mesenchymal stem cells to differentiate into osteoblasts. Fibroblasts and osteoblasts belong to the same lineage. Osteoclasts can induce osteoblast differentiation. Therefore, we infer that osteoclasts may participate in and play an important role in the process of fibroblast mediated heterotopic ossification.
Objective: the purpose of this study is to understand the interaction of human peripheral blood mononuclear cells from osteoclast like cells and human ligamentous fibroblasts in vitro. This topic focuses on (1) the study of osteoclast induction for the mineralization of human ligamentous fibroblasts and preliminary exploration of its mechanism of action; (2) observe human toughening Can fibroblast conditioned medium induce osteoclast precursors to osteoclast differentiation?
Contents and methods: isolated peripheral blood mononuclear cells were separated by Ficoll-Paque density gradient centrifugation, and CD14~+ mononuclear cells were separated by immunomagnetic beads; CD14~+ cells were induced to differentiate into osteoclast like cells by two factors of M-CSF and RANKL; the morphological observation, tartarate acid phosphatase (TRAP) staining and scanning electron microscopy were used to observe bone absorption. The characteristics of the osteoclast were fixed. At the same time, the fibroblasts were cultured in the human spinal ligament with collagenase digestion. The osteoclast conditioned medium (Osteoclasts conditioned medium) was used to treat the ligament fibroblasts and the BCIP/NBT alkaline phosphatase chromogenic kit was used to detect the expression of fibrinolytic alkaline phosphatase (ALP), and the alizarin red staining view The mineralized nodules were observed and the mineralization level was evaluated. The expression profiles of mRNA and microRNA (miRNA) in fibroblasts were detected by mRNA expression chip and microRNA chip, and the bioinformatics analysis, such as GO analysis and pathway analysis of differentially expressed genes, was used to make use of another patient's toughening. The fibroblasts cultured in the Central Plains, the repeated conditioned medium and the chip experiment. The osteoclast precursor was cultured in the conditioned medium of fibroblasts, the CD14~+ mononuclear cells were cultured. The positive rate of TRAP was detected by the differentiation.TRAP staining kit of osteoclasts, and the expression of the osteoclast differentiation marker gene was detected by RT-PCR.
Results: the positive rate of alkaline phosphatase expression in fibroblasts cultured with osteoclast cell conditioned medium was significantly higher than that of the control group. Alizarin red staining observed obvious mineralized nodules. Compared with the control group, the expression of mRNA and miRNA were significantly different from the.MRNA expression spectrum chip detection results, 522 genes were up and 415 genes were down regulated. More than two times, miRNA chip results, 28 miRNA up, 59 miRNA down 2 times. Repeat experiment mRNA chip detection results: 3707 mRNA up, 3251 mRNA downregulation, microRNA chip repeat results: 23 miRNA up, 36 miRNA downregulation.
CD14~+ mononuclear cells treated with fibroblast conditioned medium had a positive rate of 40% TRAP, and the expression of TRAP and other osteoclast differentiation markers were detected by RT-PCR.
Conclusion: osteoclast like cells can induce the mineralization of ligamentous fibroblasts in vitro, and miRNA participates in the regulation of expression in this process. At the same time, the conditioned medium of fibroblasts can induce the differentiation of CD14~+ cells to osteoclasts, and the interaction between fibroblasts and osteoclasts in the process of metabolism and differentiation.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329.2

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