本文選題：SDF-1/CXCR4信號(hào)通路 + AMD3100　； 參考：《濟(jì)南大學(xué)》2012年碩士論文

【摘要】：SDF-1（stromal-derived factor-1，間質(zhì)衍生因子），，也被稱為CXCL12，通過激活G蛋白偶聯(lián)受體——CXCR4（C-X-C chemokine receptor4，C-X-C型趨化因子受體4），在調(diào)節(jié)干細(xì)胞/前體細(xì)胞及淋巴細(xì)胞運(yùn)輸、炎癥反應(yīng)、免疫系統(tǒng)的維持、紅細(xì)胞生成、心臟及血管等器官發(fā)生以及組織器官再生等過程中起著重要作用。 AMD3100是一種人工合成的小分子大環(huán)類SDF-1受體CXCR4特異性拮抗劑，可有效地與CXCR4結(jié)合，阻斷SDF-1與CXCR4之間的結(jié)合以及隨后的信號(hào)轉(zhuǎn)導(dǎo)，可作為治療非何杰金氏淋巴瘤（NML）及多發(fā)性骨髓瘤（NM）的一種有效的造血干細(xì)胞動(dòng)員劑。研究表明，AMD3100在SDF-1/CXCR4信號(hào)通路參與調(diào)控的病理過程中發(fā)揮重要作用。 最近研究表明，SDF-1/CXCR4信號(hào)通路在骨生長分化及重塑、鈣動(dòng)員、骨髓成髓作用以及成骨細(xì)胞分化過程中起作用，Kitaori等人研究表明SDF-1/CXCR4信號(hào)通路在體內(nèi)骨骼修復(fù)過程中參與骨折部位間充質(zhì)干細(xì)胞（mesenchymal stem cells, MSCs）的募集；Zhu及Hosogane等人研究表明該通路參與骨形成蛋白2（bone morphogenetic protein2, BMP2）介導(dǎo)的初級(jí)MSC或MSC細(xì)胞系的成骨分化過程。并且，在多種干細(xì)胞以及前體細(xì)胞中均有SDF-1和CXCR4的表達(dá)，其中骨髓間充質(zhì)干細(xì)胞中二者表達(dá)尤為豐富，一旦細(xì)胞開始分化二者表達(dá)量會(huì)顯著下降，Kortesidis A等在體外實(shí)驗(yàn)中發(fā)現(xiàn)SDF-1和CXCR4在前成骨細(xì)胞中高度表達(dá)，而在成熟的成骨細(xì)胞、破骨細(xì)胞中含量較低，這表明SDF-1/CXCR4信號(hào)通路在成骨分化前期起著重要作用。因此，成骨分化過程中SDF-1/CXCR4信號(hào)通路拮抗劑AMD3100的有效性及毒性研究顯得尤為必要。到目前為止，有關(guān)AMD3100在MSC細(xì)胞成骨分化初期發(fā)揮作用的研究甚少，尤其是AMD3100對(duì)前成骨細(xì)胞基質(zhì)礦化調(diào)節(jié)的研究尚未見文獻(xiàn)報(bào)道。 另外，SDF-1與CXCR4的結(jié)合會(huì)激活JAK/STAT通路， JAK（Janus kinase,Janus激酶）被CXCR4激活并與之結(jié)合，該結(jié)合可促進(jìn)轉(zhuǎn)錄因子STAT3（signaltransducer and activator of transcription3,信號(hào)傳導(dǎo)子及轉(zhuǎn)錄激活子3）的募集和酪氨酸磷酸化，而AMD3100可能通過調(diào)節(jié)STAT3而在前成骨細(xì)胞的成骨分化過程中發(fā)揮一定的作用。 目的：本研究旨在闡釋SDF-1/CXCR4通路在成骨分化過程中的具體作用機(jī)制，便于更深入、系統(tǒng)地研究和了解成骨分化復(fù)雜的信號(hào)通路網(wǎng)絡(luò)。采用CXCR4拮抗劑AMD3100抑制SDF-1和CXCR4的結(jié)合，為進(jìn)一步研究細(xì)胞成骨分化調(diào)控過程中SDF-1/CXCR4信號(hào)通路抑制劑的作用機(jī)制提供新思路。 方法：本研究擬采用小鼠顱頂前骨細(xì)胞系——MC3T3-E1亞克隆14，觀察CXCR4拮抗劑AMD3100對(duì)地塞米松(dexamethasone, DEX)、抗壞血酸(L-ascorbic acid, AA)和β-磷酸甘油(β-glycerophosphate, β-GP)誘導(dǎo)的MC3T3-E1細(xì)胞成骨分化的影響。本研究擬采用堿性磷酸酶（alkaline phosphatase，ALP）活性檢測的方法檢測礦化前后ALP活性變化，采用茜素紅染色法觀察礦化結(jié)節(jié)的形成，并應(yīng)用熒光定量PCR的方法檢測礦化標(biāo)志蛋白ALP、Runx2/cbfal、ANK、OCN等基因的表達(dá)，以及采用Western Blot的方法檢測SDF-1/CXCR4通路下游蛋白STAT3和Phospho-STAT3的表達(dá)，用上述方法證實(shí)MC3T3-E1細(xì)胞礦化過程以及AMD3100抑制劑對(duì)礦化的影響情況。本實(shí)驗(yàn)通過觀察細(xì)胞各時(shí)相成骨分化的變化，研究SDF-1/CXCR4通路及其下游蛋白STAT3在成骨分化過程中所發(fā)揮的作用。 結(jié)果：（1）ALP活性檢測實(shí)驗(yàn)結(jié)果表明，采用地塞米松、抗壞血酸及β-磷酸甘油誘導(dǎo)的MC3T3-E1細(xì)胞在經(jīng)CXCR4特異性抑制劑AMD3100處理之后，在細(xì)胞成骨分化的第3、6、9天，與正常對(duì)照組相比，礦化組ALP活性整體呈升高的趨勢，且在第6天出現(xiàn)ALP活性峰值；相應(yīng)的抑制劑組ALP活性均呈降低趨勢，且在成骨分化第6天不同濃度的抑制劑組出現(xiàn)AMD3100劑量依賴趨勢。（2）茜素紅染色實(shí)驗(yàn)發(fā)現(xiàn)，MC3T3-E1細(xì)胞在地塞米松、抗壞血酸和β-磷酸甘油介導(dǎo)的礦化誘導(dǎo)14、21天之后已能表達(dá)成骨細(xì)胞表型。但是50μM及100μM的兩組抑制劑組均未見有統(tǒng)計(jì)學(xué)意義的茜素紅染色程度的減弱。（3）熒光定量PCR的結(jié)果表明，在MC3T3-E1細(xì)胞礦化誘導(dǎo)第6、9天，細(xì)胞成骨分化早期標(biāo)志性基因ALP、Runx2以及末期標(biāo)志性基因ANK、OCN的表達(dá)在地塞米松、抗壞血酸和β-磷酸甘油介導(dǎo)的礦化誘導(dǎo)作用下均出現(xiàn)上調(diào)趨勢，相比于礦化組，在AMD3100作用下，抑制劑組均出現(xiàn)相應(yīng)的下調(diào)趨勢。（4）Western Blot結(jié)果表明，在MC3T3-E1細(xì)胞礦化誘導(dǎo)第6天，在地塞米松、抗壞血酸和β-磷酸甘油介導(dǎo)的礦化誘導(dǎo)作用下Phospho-STAT3表達(dá)量升高，而在AMD3100作用下Phospho-STAT3表達(dá)量出現(xiàn)明顯下調(diào)趨勢。 結(jié)論：本研究表明CXCR4抑制劑AMD3100對(duì)MC3T3-E1細(xì)胞成骨分化起到一定的調(diào)節(jié)作用，AMD3100在細(xì)胞成骨分化早期下調(diào)ALP活性，但在晚期并不能抑制MC3T3-E1細(xì)胞的基質(zhì)礦化，這也可能與SDF-1/CXCR4信號(hào)通路在細(xì)胞分化前期發(fā)揮作用有關(guān)。細(xì)胞成骨分化早期標(biāo)志性基因ALP、Runx2以及末期標(biāo)志性基因ANK、OCN的mRNA表達(dá)在AMD3100的作用下出現(xiàn)下調(diào)，并且AMD3100在成骨分化早期抑制STAT3的磷酸化，這似乎提示SDF-1/CXCR4通路在成骨分化早期通過STAT3蛋白磷酸化發(fā)揮部分調(diào)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.
【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Inhibition of CXCR4 activity with AMD3100 decreases invasion of human colorectal cancer cells in vitro[J];World Journal of Gastroenterology;2008年15期

本文編號(hào)：2043007