MIP-1α和sclerostin在骨髓瘤骨病患者中的表达及相互关系的研究

发布时间：2018-04-28 12:02

本文选题：多发性骨髓瘤 + 骨髓瘤骨病　；参考：《广西医科大学》2016年博士论文

【摘要】：骨髓瘤骨病(MBD)是MM患者的最常见的并发症之一。MBD发生的主要机制:在骨髓微环境中,瘤细胞与骨髓基质细胞(BMSCs)通过RANKL/RANK等经典途径、巨噬细胞炎症蛋白-1α(MIP-1α)等非经典途径的信号通路,增强破骨细胞(OC)的数量、抑制成骨细胞(OB)的功能,最后导致溶骨性损害。本人前期研究及国内外最近研究报道MIP-1α不仅有增加OC的数量和增强OC的功能,还有抑制OB的分化和成熟,但抑制OB的具体机制不明。骨硬化蛋白(sclerostin)是SOST基因的表达产物,参与骨形成调节,决定骨重建过程中的骨量和结构,是Wnt/β-信号通路的重要抑制剂,而Wnt/β-信号通路在介导OB分化、成熟及功能调节中是非常重要的。最近研究报道:MM患者外周血sclerostin增高,但其增多的机制以及与MM患者的生物学特征尚不明确。本研究试图通过探讨MBD患者骨髓血浆及骨髓瘤细胞株MIP-1α和sclerostin蛋白的表达,以此探明这两种蛋白与患者的临东快氮分期、生化特征、预后及两者之间的关系等。通过从美国国立生物技术信息中心(GEO)的共享数据库中下载MBD相关的基因芯片表达数据谱GSE754和GSE755,运用生物信息学相关工具进行数据和文献挖掘,通过大数据分析MIP-1α和sclerostin蛋白是否具有一定的关联性。进一步在体外验证这两种蛋白的相互关系,为MBD患者的防治开拓新思路和提供理论依据。本论文主要从如下三方面进行探讨。第一部分MIP和sclerostin在骨髓瘤骨病患者中的表达及与临东目的:本研究旨在探讨MIP-1α和sclerostin在MBD中的表达,MIP-1α和sclerostin与MBD分级、ISS临东及两者相互之间的关系。方法:以53名MBD患者(其中女24名,男29名),人多发性骨髓瘤细胞株RPMI-8226、H929及30名白细胞减少症、免疫性血小板减少症患者为研究对象。酶联免疫吸附法(ELISA)定量检测骨髓血浆MIP-1α和sclerostin蛋白的水平;RT-PCR技术检测MIP-1α和sclerostin蛋白的m RNA表达;电化学发光免疫法测量甲状旁腺素(PTH)和1,25(OH)2Vit D3的水平,全自动生化分析仪检测患者的各项常规生化指标等。实验数据以均数±标准差表示,使用SPSS18.0统计学软件进行分析。结果:27例患者的sclerostin水平升高,37例患者中MIP-1α蛋白的水平明显升高。MBD患者的MIP-1α(153.35±81.09 pg/ml)和sclerostin(539.42±201.86 pg/ml)蛋白浓度要高于对照组(t=6.85和8.26,p均为0.00)。体外骨髓瘤细胞株RPMI-8226、H929培养上清液中MIP-1α和sclerostin蛋白的水平与空白对照组相比均明显增高(t值分别为23.52,13.42;P值均为0.000.01),RT-PCR的结果分析也证实了大部分的MBD患者骨髓单个核细胞(BMMNCs)及两种骨髓瘤细胞株均表达这两种蛋白,但表达的量不一致。临抗ISS分期3期患者中的MIP-1α(173.81±97.74 pg/ml)和sclerostin蛋白(0.61±0.20ng/ml)的表达水平明显高于2期的患者(MIP-1α:117.73±54.35 pg/ml,sclerostin:0.42±0.18 ng/ml)(p值分别为0.01、0.06);MBD分级C组患者的MIP-1α蛋白(MIP-1α170.37±103.69 pg/ml)和sclerostin(0.60±0.02 pg/ml)水平也明显高于A+B组(MIP-1α:119.82±38.15 pg/ml,sclerostin:0.45±0.19 ng/ml))(P值均小于0.01)。sclerostin水平与MIP-1α蛋白(r=0.720,p=0.000.001),β2-MG(r=0.467,p=0.000.001,LDH(r=0.453,p=0.001),a Ca水平(r=0.313,p=0.023)之间呈明显的正相关,而sclerostin蛋白与b ALP(r=-0.578,p=0.000.001),HB(r=-0.412,p=0.002),ALB(r=-0.388,p=0.004)之间呈负相关;sclerostin、MIP-1α蛋白的水平与MBD分级(r=0.439和0.358,p=0.001和0.008)和ISS分期(r=0.436和0.323,p=0.001、0.018)呈正相关,高水平(0.54ng/ml)的sclerostin蛋白的MBD患者的中位生存时间也要短于低水平的患者(X2=7.376,p=0.007),而高水平(153.71pg/ml)的MIP-1α蛋白的MBD患者的中位生存时间虽短于低水平的患者,但两者比较无统计学差异(χ2=2.94,p=0.086)。双重线性回归结果提示ISS分期、sclerostin蛋白,β2-MG,MIP-1α蛋白是MBD分级独立的预后不良因素。结论:1.MIP-1α、sclerostin在MBD患者骨髓中水平增高、骨髓瘤细胞系能分泌这两种蛋白,但不同细胞系表达水平不一定相同;2.MIP-1α、sclerostin与临床ISS分期、MBD分级、与反应瘤负荷的相关指标β2-MG、LDH水平呈正相关,而与b ALP、HB、ALB则呈负相关。3.高水平(0.54ng/ml)的sclerostin的MBD患者的中位生存时间要短于低水平的MBD患者.4.MIP-1α与sclerostin呈明显的正相关。目的:采用生物信息学各种软件和在线工具进行筛选MBD的相关基因,并对其进行数据和文献挖掘,进一步通过大数据了解MIP-1α与sclerostin是否具有一定的关联性。方法:以MBD相关的两组患者基因芯片表达数据谱GSE754(139例有骨质破坏的MM患者)和GSE755(34例无骨质破坏的患者)为研究材料,运用Gene Spring GX11.5软件对基因芯片数据进行差异基因分析,GATHER等分析工具注释MBD相关差异基因可能的分子功能、参与的各种信号通路及通路富集,Cytosine软件分析差异基因编码蛋白质之间的相互作用关系。结果:从GSE754和GSE755基因芯片数据中分析得到共173个样本,经Genesis软件分析得出435个共同表达的差异基因,其中高表达的有286个基因,低表达的有149个基因。导入GATHER后发现这些差异基因主要与细胞增殖、钙磷代谢、细胞周期,细胞粘附与迁移、细胞凋亡、蛋白酶体、MAPK、趋化因子信号通路、Erb B、细胞周期,细胞减速分裂、Wnt、T、B细胞信号通路、JakSTAT、Rho A/ROCK等信号通路有关。将差异基因导入Cytosine软件中分析,结果发现BMD相关基因编码的蛋白质间的相互联系主要集中在NF-κB,PTK2B,ADD3,SPP1,IGF2,HSF2,HSF2,NTRK2,ADD3,GST6、CCL3、IL-6,Rh OA、sclerostin等基因上。结论:通过对基因芯片大数据进行生物信息学相关分析筛选得出MIP-1α与sclerostin具有一定的关联性,参与MBD的发生主要涉及到蛋白酶体、MAPK、Erb B、细胞周期,Wnt、Jak-STAT等信号通路。第二部分:骨髓瘤骨病相关基因的筛选及生物信息学分析目的:拟在体外通过细胞培养,外源性给予MIP-1α和sclerostin、MIP-1α和sclerostin单抗增加或中和骨髓瘤细胞株RPMI-8226、H929高表达sclerostin/MIP-1α的水平,观察该细胞的sclerostin/MIP-1α的水平变化。初步验证这两种蛋白的关联性。方法:以人骨髓瘤细胞系RPMI-8226、H929为研究对象,ELISA定量检测各组细胞上清液中的MIP-1α和sclerostin蛋白水平,RT-PCR定量检测各组细胞的MIP-1α和sclerostin m RNA水平。结果:两组细胞上清液中加入MIP-1α后再培养24小时,MIP-1α和sclerostin水平较不干预组明显增高(p值均为0.00);加入MIP-1α单抗后,两组细胞中的MIP-1α和sclerostin水平较不干预组明显下降(p值分别为0.00和0.001);加入sclerostin及sclerostin单抗后,两组细胞的MIP-1α水平较不干预组改变不明显(p值均大于0.05),加入sclerostin后,两组细胞的sclerostin水平明显增高(p值均小于0.01),而加入sclerostin单抗后,两组细胞的sclerostin水平明显下降(p值均小于0.01)。结论:MIP-1α可介导骨髓瘤细胞分泌sclerostin,具体机制需进一步研究。总之,本研究验证了sclerostin、MIP-1α两种蛋白在大部分MBD患者骨髓中及骨髓瘤细胞株中呈高表达,且与瘤负荷、MBD分级、ISS分期相关,两种蛋白呈明显的正相关。通过从GEO下载基因芯片大数据,运用生物信息学相关软件和在线工具进一步验证了sclerostin、MIP-1α两种蛋白有一定的关联。在体外通过细胞培养技术验证了MIP-1α可能通过各种途径促进瘤细胞或骨髓微环境分泌sclerostin。这揭示了MIP-1α在MBD中抑制OB的功能可能是通过sclerostin来介导的可能机制。第三部分体外通过细胞培养验证MIP-1α和sclerostin的关系
[Abstract]:Myeloma osteopathy (MBD) is the main mechanism of one of the most common complications of MM patients: in the bone marrow microenvironment, tumor cells and bone marrow stromal cells (BMSCs) can enhance the number of osteoclast (OC) and inhibit osteoblast through the classical pathway of RANKL/RANK, such as RANKL/RANK and other classical pathways, such as macrophage inflammatory protein -1 alpha (MIP-1 alpha). The function of (OB), which eventually leads to osteolytic damage. My previous studies and recent studies at home and abroad have reported that MIP-1 alpha not only increases the number of OC and enhances the function of OC, but also inhibits the differentiation and maturation of OB, but the specific mechanism of the inhibition of OB is unknown. The bone sclerosis protein (sclerostin) is the expression product of the SOST gene, participates in the regulation of bone formation, and determines the bone. Bone mass and structure during the reconstruction are important inhibitors of the Wnt/ beta signaling pathway, and the Wnt/ beta signaling pathway is very important in mediating OB differentiation, maturation and functional regulation. Recent studies have reported that the peripheral blood sclerostin of MM patients is higher, but the mechanism of its increase and the biological characteristics of the patients with MM are not yet clear. This study attempts to use this study. To explore the expression of MIP-1 alpha and sclerostin protein in the bone marrow plasma and myeloma cell lines of MBD patients, in order to identify the two proteins and the patient's Near East fast nitrogen staging, biochemical characteristics, prognosis and the relationship between the two. The MBD related gene chip expression was downloaded from the shared database of the National Biotechnology Information Center (GEO) of the United States. Data spectrum GSE754 and GSE755, using bioinformatics related tools to carry out data and literature mining, through large data analysis whether MIP-1 alpha and sclerostin protein have a certain relevance. Further to verify the relationship between these two proteins in vitro, to open up new ideas and theoretical basis for the prevention and control of MBD patients. The following three aspects are discussed. Part 1: expression of MIP and sclerostin in patients with myeloma bone disease and its immediate East purpose: This study aims to explore the expression of MIP-1 alpha and sclerostin in MBD, MIP-1 alpha and sclerostin and MBD grading, ISS near the East and the relationship between the two. Methods: 53 MBD patients (24 women, 29 men), and more people. Hair myeloma cell lines RPMI-8226, H929 and 30 leukenia and immunological thrombocytopenia were studied. Enzyme linked immunosorbent assay (ELISA) was used to detect the level of MIP-1 alpha and sclerostin in bone marrow plasma; RT-PCR technique was used to detect the m RNA expression of MIP-1 A and sclerostin egg white; electrochemiluminescence immunoassay was used to measure the thyroid gland. The levels of parathyroid (PTH) and 1,25 (OH) 2Vit D3 were measured by an automatic biochemical analyzer. The experimental data were expressed with mean standard deviation, and the SPSS18.0 statistics software was used for analysis. Results: the level of sclerostin in 27 patients was elevated, and the level of MIP-1 alpha protein in 37 patients significantly increased MIP- in.MBD patients. The concentration of 1 alpha (153.35 + 81.09 pg/ml) and sclerostin (539.42 + 201.86 pg/ml) protein was higher than that in the control group (t=6.85 and 8.26, P was 0). The level of MIP-1 A and sclerostin protein in the culture supernatant of H929 was compared with that of the blank control group (t value was 23.52,13.42; P values were all), The results also showed that most of the MBD patients' bone marrow mononuclear cells (BMMNCs) and two myeloma cell lines expressed these two proteins, but the amount of expression was not consistent. The expression level of MIP-1 alpha (173.81 + 97.74 pg/ml) and sclerostin protein (0.61 + 0.20ng/ml) in the 3 stage patients with ISS staging was significantly higher than that of the 2 stage patients (MIP-1 A: 1). 17.73 + 54.35 pg/ml, sclerostin:0.42 + 0.18 ng/ml) (P value was 0.01,0.06), MIP-1 alpha protein (MIP-1 alpha 170.37 + 103.69 pg/ml) and sclerostin (0.60 + 0.02 pg/ml) in MBD graded C group were also significantly higher than that of the group (119.82 + 38.15, 0.19). R=0.720 (p=0.000.001), beta 2-MG (r=0.467, p=0.000.001, LDH (r=0.453, p=0.001), a Ca level (r=0.313, p=0.023) showed a negative correlation; .358, p=0.001, and 0.008) were positively correlated with ISS staging (r=0.436 and 0.323, p=0.001,0.018). The median survival time of the MBD patients with high level (0.54ng/ml) sclerostin protein was also shorter than that of low level patients (X2=7.376, p=0.007), while the median survival time of the high level (153.71pg/ml) MIP-1 alpha protein was shorter than the low level. But there was no statistical difference between the two (x 2=2.94, p=0.086). The results of double linear regression suggested that ISS staging, sclerostin protein, beta 2-MG, MIP-1 alpha protein were independent prognostic factors of MBD classification. Conclusion: 1.MIP-1 a, sclerostin in the bone marrow of MBD patients, the two proteins can be secreted by the bone myeloma cell line, but the expression of different cell lines is expressed. The level of 2.MIP-1 alpha, sclerostin and clinical ISS staging and MBD grading were positively correlated with the level of the response to the response to the tumor load, the level of LDH was positively correlated with the B ALP, HB, ALB, and the median survival time of the patients was shorter than the low level. Objective: to select the related genes of MBD by using various bioinformatics software and online tools, and to carry out data and literature mining, and to further understand whether there is a certain correlation between MIP-1 alpha and sclerostin through large data. Methods: the data spectrum GSE754 (139 cases of bone) was expressed by two groups of MBD related gene chips. MM patients with qualitative damage) and GSE755 (34 patients without bone destruction) were used as research materials. Gene Spring GX11.5 software was used to carry out differential gene analysis on gene chip data. GATHER and other analysis tools annotate the possible molecular functions of MBD related differentially genes, various signaling pathways and pathways involved in the genetic analysis, and Cytosine software to analyze differential genes. The interaction relationship between encoding proteins. Results: a total of 173 samples were obtained from GSE754 and GSE755 gene chip data, and 435 differentially expressed genes were analyzed by Genesis software, of which 286 genes were expressed in high expression and 149 genes were low in expression. After introducing GATHER, these differentially expressed genes were found to increase mainly with cells. Colonization, calcium and phosphorus metabolism, cell cycle, cell adhesion and migration, cell apoptosis, proteasome, MAPK, chemokine signaling pathway, Erb B, cell cycle, cell deceleration and division, Wnt, T, B cell signaling pathways, JakSTAT, Rho A/ROCK and other signaling pathways. Analysis of differential genes into Cytosine software, and the results of proteins encoded by BMD related genes The interrelation between quality is mainly focused on NF- kappa B, PTK2B, ADD3, SPP1, IGF2, HSF2, HSF2, NTRK2, ADD3, GST6, CCL3, etc. Body, MAPK, Erb B, cell cycle, Wnt, Jak-STAT and other signaling pathways. The second part: screening and bioinformatics analysis of myeloma bone disease related genes and bioinformatics analysis: to be cultured in vitro by cell culture, exogenous to MIP-1 alpha and sclerostin, MIP-1 A and sclerostin McAbs to increase or neutralize myeloma cell strain RPMI-8226, H929 high expression sclerostin/MIP The level of -1 alpha and the level of sclerostin/MIP-1 alpha in the cell were observed. The correlation of the two proteins was preliminarily verified. Methods: the human myeloma cell line RPMI-8226, H929 was used as the research object. MIP-1 alpha and sclerostin protein levels in the cell supernatant were detected by ELISA. MIP-1 alpha and sclerostin M were detected by RT-PCR. RNA level. Results: after adding MIP-1 alpha in the two groups of cell supernatants for 24 hours, the level of MIP-1 alpha and sclerostin was significantly higher than that in the non intervention group (P value was 0). After adding MIP-1 a monoclonal antibody, the levels of MIP-1 alpha and sclerostin in the two groups were significantly lower than those in the non intervention group (P values were 0 and 0.001 respectively), and sclerostin and sclerostin were added. After mAb, the level of MIP-1 alpha in the two groups was not significantly higher than that in the non intervention group (P value was greater than 0.05). After adding sclerostin, the level of sclerostin in the two groups increased significantly (P value was less than 0.01), and the sclerostin level of the two groups was significantly decreased after adding sclerostin MAb (P value was less than 0.01). Conclusion: MIP-1 alpha mediates myeloma fine. Cell secretion of sclerostin, specific mechanisms need to be further studied. In conclusion, this study confirmed that sclerostin, MIP-1 alpha two proteins are highly expressed in most of MBD patients' bone marrow and myeloma cell lines, and are associated with tumor load, MBD classification, ISS staging, and a significant positive correlation. Information science related software and online tools further verified the association between sclerostin, MIP-1 alpha and two proteins. Through cell culture in vitro, MIP-1 alpha may promote tumor cells or bone marrow microenvironment to secrete sclerostin.. This reveals that the function of MIP-1 a in the suppression of OB in MBD may be through sclerostin The third part is to verify the relationship between MIP-1 and sclerostin in vitro.

【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.3

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