CXCR4抑制剂AMD3100对MC3T3-E1细胞成骨分化的影响

发布时间：2018-06-20 04:43

本文选题：SDF-1/CXCR4信号通路 + AMD3100　；参考：《济南大学》2012年硕士论文

【摘要】：SDF-1（stromal-derived factor-1，间质衍生因子），，也被称为CXCL12，通过激活G蛋白偶联受体——CXCR4（C-X-C chemokine receptor4，C-X-C型趋化因子受体4），在调节干细胞/前体细胞及淋巴细胞运输、炎症反应、免疫系统的维持、红细胞生成、心脏及血管等器官发生以及组织器官再生等过程中起着重要作用。 AMD3100是一种人工合成的小分子大环类SDF-1受体CXCR4特异性拮抗剂，可有效地与CXCR4结合，阻断SDF-1与CXCR4之间的结合以及随后的信号转导，可作为治疗非何杰金氏淋巴瘤（NML）及多发性骨髓瘤（NM）的一种有效的造血干细胞动员剂。研究表明，AMD3100在SDF-1/CXCR4信号通路参与调控的病理过程中发挥重要作用。最近研究表明，SDF-1/CXCR4信号通路在骨生长分化及重塑、钙动员、骨髓成髓作用以及成骨细胞分化过程中起作用，Kitaori等人研究表明SDF-1/CXCR4信号通路在体内骨骼修复过程中参与骨折部位间充质干细胞（mesenchymal stem cells, MSCs）的募集；Zhu及Hosogane等人研究表明该通路参与骨形成蛋白2（bone morphogenetic protein2, BMP2）介导的初级MSC或MSC细胞系的成骨分化过程。并且，在多种干细胞以及前体细胞中均有SDF-1和CXCR4的表达，其中骨髓间充质干细胞中二者表达尤为丰富，一旦细胞开始分化二者表达量会显著下降，Kortesidis A等在体外实验中发现SDF-1和CXCR4在前成骨细胞中高度表达，而在成熟的成骨细胞、破骨细胞中含量较低，这表明SDF-1/CXCR4信号通路在成骨分化前期起着重要作用。因此，成骨分化过程中SDF-1/CXCR4信号通路拮抗剂AMD3100的有效性及毒性研究显得尤为必要。到目前为止，有关AMD3100在MSC细胞成骨分化初期发挥作用的研究甚少，尤其是AMD3100对前成骨细胞基质矿化调节的研究尚未见文献报道。另外，SDF-1与CXCR4的结合会激活JAK/STAT通路， JAK（Janus kinase,Janus激酶）被CXCR4激活并与之结合，该结合可促进转录因子STAT3（signaltransducer and activator of transcription3,信号传导子及转录激活子3）的募集和酪氨酸磷酸化，而AMD3100可能通过调节STAT3而在前成骨细胞的成骨分化过程中发挥一定的作用。目的：本研究旨在阐释SDF-1/CXCR4通路在成骨分化过程中的具体作用机制，便于更深入、系统地研究和了解成骨分化复杂的信号通路网络。采用CXCR4拮抗剂AMD3100抑制SDF-1和CXCR4的结合，为进一步研究细胞成骨分化调控过程中SDF-1/CXCR4信号通路抑制剂的作用机制提供新思路。方法：本研究拟采用小鼠颅顶前骨细胞系——MC3T3-E1亚克隆14，观察CXCR4拮抗剂AMD3100对地塞米松(dexamethasone, DEX)、抗坏血酸(L-ascorbic acid, AA)和β-磷酸甘油(β-glycerophosphate, β-GP)诱导的MC3T3-E1细胞成骨分化的影响。本研究拟采用碱性磷酸酶（alkaline phosphatase，ALP）活性检测的方法检测矿化前后ALP活性变化，采用茜素红染色法观察矿化结节的形成，并应用荧光定量PCR的方法检测矿化标志蛋白ALP、Runx2/cbfal、ANK、OCN等基因的表达，以及采用Western Blot的方法检测SDF-1/CXCR4通路下游蛋白STAT3和Phospho-STAT3的表达，用上述方法证实MC3T3-E1细胞矿化过程以及AMD3100抑制剂对矿化的影响情况。本实验通过观察细胞各时相成骨分化的变化，研究SDF-1/CXCR4通路及其下游蛋白STAT3在成骨分化过程中所发挥的作用。结果：（1）ALP活性检测实验结果表明，采用地塞米松、抗坏血酸及β-磷酸甘油诱导的MC3T3-E1细胞在经CXCR4特异性抑制剂AMD3100处理之后，在细胞成骨分化的第3、6、9天，与正常对照组相比，矿化组ALP活性整体呈升高的趋势，且在第6天出现ALP活性峰值；相应的抑制剂组ALP活性均呈降低趋势，且在成骨分化第6天不同浓度的抑制剂组出现AMD3100剂量依赖趋势。（2）茜素红染色实验发现，MC3T3-E1细胞在地塞米松、抗坏血酸和β-磷酸甘油介导的矿化诱导14、21天之后已能表达成骨细胞表型。但是50μM及100μM的两组抑制剂组均未见有统计学意义的茜素红染色程度的减弱。（3）荧光定量PCR的结果表明，在MC3T3-E1细胞矿化诱导第6、9天，细胞成骨分化早期标志性基因ALP、Runx2以及末期标志性基因ANK、OCN的表达在地塞米松、抗坏血酸和β-磷酸甘油介导的矿化诱导作用下均出现上调趋势，相比于矿化组，在AMD3100作用下，抑制剂组均出现相应的下调趋势。（4）Western Blot结果表明，在MC3T3-E1细胞矿化诱导第6天，在地塞米松、抗坏血酸和β-磷酸甘油介导的矿化诱导作用下Phospho-STAT3表达量升高，而在AMD3100作用下Phospho-STAT3表达量出现明显下调趋势。结论：本研究表明CXCR4抑制剂AMD3100对MC3T3-E1细胞成骨分化起到一定的调节作用，AMD3100在细胞成骨分化早期下调ALP活性，但在晚期并不能抑制MC3T3-E1细胞的基质矿化，这也可能与SDF-1/CXCR4信号通路在细胞分化前期发挥作用有关。细胞成骨分化早期标志性基因ALP、Runx2以及末期标志性基因ANK、OCN的mRNA表达在AMD3100的作用下出现下调，并且AMD3100在成骨分化早期抑制STAT3的磷酸化，这似乎提示SDF-1/CXCR4通路在成骨分化早期通过STAT3蛋白磷酸化发挥部分调控作用。
[Abstract]:SDF-1 (stromal-derived factor-1, interstitial derived factor), also known as CXCL12, activates the G protein coupled receptor, CXCR4 (C-X-C chemokine receptor4, C-X-C chemokine receptor 4), to regulate the transport of stem cell / precursor cells and lymphocytes, inflammation, maintenance of the immune system, erythrocytic, cardiac and vascular organs. It plays an important role in the process of tissue regeneration.
AMD3100 is a synthetic small molecule macrocyclic SDF-1 receptor CXCR4 specific antagonist. It can effectively bind to CXCR4, block the combination of SDF-1 and CXCR4 and subsequent signal transduction. It can be used as an effective hematopoietic stem cell mobilization agent for the treatment of non Hodgkin's lymphoma (NML) and multiple myeloma (NM). MD3100 plays an important role in the pathological process of SDF-1/CXCR4 signaling pathway.
Recent studies have shown that SDF-1/CXCR4 signaling pathway plays a role in bone growth differentiation and remodeling, calcium mobilization, marrow myelogenesis, and osteoblast differentiation. Kitaori et al. Studies show that SDF-1/CXCR4 signaling pathway participates in the recruitment of bone mesenchymal stem cells (mesenchymal stem cells, MSCs) during bone repair in vivo; Zh U and Hosogane studies have shown that the pathway participates in the osteogenic differentiation of the primary MSC or MSC cell lines mediated by bone morphogenetic protein 2 (bone morphogenetic protein2, BMP2), and the expression of SDF-1 and CXCR4 in a variety of stem cells and precursor cells, especially in the two bone marrow mesenchymal stem cells, once the cells are opened. The expression of two of the two first differentiation would be significantly decreased, and the high expression of SDF-1 and CXCR4 in the anterior osteoblasts in vitro, and the low content of the mature osteoblasts and osteoclasts in the mature osteoblasts, indicating that the SDF-1/CXCR4 signaling pathway plays an important role in the prophase of osteogenesis. Therefore, the SDF-1/CXCR4 signal in the process of osteogenesis differentiation. The study of the effectiveness and toxicity of the pathway antagonist AMD3100 is particularly necessary. So far, little research has been made about the role of AMD3100 in the early differentiation of MSC cells, especially the study on the regulation of the mineralization of the precursor osteoblasts by AMD3100 has not yet been reported.
In addition, the combination of SDF-1 and CXCR4 activates the JAK/STAT pathway, and JAK (Janus kinase, Janus kinase) is activated and combined with CXCR4, which can promote the recruitment and tyrosine phosphorylation of the transcription factor STAT3 (signaltransducer and activator), signal transduction and transcriptional activator 3. It plays a role in osteoblastic differentiation of osteoblasts.
Objective: the purpose of this study is to explain the specific mechanism of SDF-1/CXCR4 pathway in the process of osteogenic differentiation in order to make it easier to study and understand the complex signaling pathway network of osteogenic differentiation. The combination of CXCR4 antagonist AMD3100 inhibits SDF-1 and CXCR4, so as to further study the SDF-1/CXCR4 signal in the process of cell osteogenesis regulation The mechanism of the pathway inhibitor provides a new way of thinking.
Methods: MC3T3-E1 subclone 14 was used in this study to observe the effect of CXCR4 antagonist AMD3100 on osteogenic differentiation induced by dexamethasone (dexamethasone, DEX), ascorbic acid (L-ascorbic acid, AA) and beta glycerol (beta -glycerophosphate, beta -GP). This study was to use alkaline phosphoric acid. The enzyme (alkaline phosphatase, ALP) activity detection method was used to detect the changes of ALP activity before and after mineralization. Alizarin red staining was used to observe the formation of mineralized nodules. The expression of mineralized marker protein ALP, Runx2/cbfal, ANK, OCN and other genes were detected by the method of fluorescence quantitative PCR, and the downstream of SDF-1/CXCR4 pathway was detected by Western Blot. The expression of protein STAT3 and Phospho-STAT3 demonstrated the mineralization process of MC3T3-E1 cells and the effect of AMD3100 inhibitor on mineralization. The effect of the SDF-1/CXCR4 pathway and its downstream protein STAT3 on the osteogenic differentiation was investigated by observing the changes of bone differentiation in each phase of the cells.
Results: (1) the results of ALP activity test showed that the MC3T3-E1 cells induced by dexamethasone, ascorbic acid and beta glycerol were treated with CXCR4 specific inhibitor AMD3100, and the ALP activity in the mineralization group was higher than that of the normal control group on the 3,6,9 day of the cell osteogenesis, and the ALP activity appeared on the sixth day. Peak value; the ALP activity of the corresponding inhibitor group was decreased, and the dose dependent trend of AMD3100 in the inhibitor group with different concentration of sixth days of osteogenesis differentiation appeared. (2) the alizarin red staining experiment found that MC3T3-E1 cells in dexamethasone, ascorbic acid and beta glycerol mediated mineralization induced 14,21 days after the induction of osteoblast phenotype. However, there was no significant reduction in the degree of alizarin red staining in 50 M and 100 M group inhibitor groups. (3) the results of fluorescence quantitative PCR showed that the early marker gene ALP, Runx2, and the end stage marker gene ANK, the expression of OCN, in dexamethasone, ascorbic acid and beta, was expressed in MC3T3-E1 cell mineralization induced 6,9 days. The upward trend of the mineralization induced by glycerol phosphate was observed. Compared with the mineralization group, the inhibitor group had a corresponding downward trend under the action of AMD3100. (4) the results of Western Blot showed that the mineralization induced by MC3T3-E1 cells was induced by dexamethasone, anti ascorbic acid and beta glycerophosphate mediated mineralization induced Phospho-STA. The expression level of T3 increased, while the expression of Phospho-STAT3 decreased significantly under the action of AMD3100.
Conclusion: This study shows that CXCR4 inhibitor AMD3100 regulates the osteogenic differentiation of MC3T3-E1 cells. AMD3100 can down regulate the activity of ALP in the early stage of osteogenic differentiation, but in the late stage it can not inhibit the matrix mineralization of MC3T3-E1 cells, which may also be related to the role of SDF-1/CXCR4 signaling pathway in the early differentiation of cells. The early differentiation marker gene ALP, Runx2 and the end-stage marker gene ANK, the mRNA expression of OCN was downregulated under the action of AMD3100, and AMD3100 inhibited STAT3 phosphorylation at the early stage of osteogenic differentiation, which seemed to suggest that the SDF-1/CXCR4 pathway partially regulates the phosphorylation of STAT3 protein in the early osteogenic differentiation.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.2

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