CN113308537A - 血清半乳糖凝集素-1作为骨代谢标志物在骨质疏松诊断中的应用 - Google Patents
血清半乳糖凝集素-1作为骨代谢标志物在骨质疏松诊断中的应用 Download PDFInfo
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Abstract
本发明涉及血清半乳糖凝集素‑1(Gal‑1)作为骨代谢标志物在骨质疏松诊断中的应用。本发明经动物实验发现小鼠血清Gal‑1呈年龄相关性下降,血清Gal‑1与小鼠股骨、胫骨和L1椎体的骨体积分数呈正相关,老年小鼠骨髓间充质干细胞和骨髓基质细胞中Gal‑1蛋白表达明显低于幼龄小鼠;经临床实验发现与年龄相关的骨密度下降的受试者血清Gal‑1水平下降,其骨密度与血清Gal‑1水平呈正相关。本发明提示Gal‑1在发现骨髓间充质干细胞衰老、早期诊断骨质疏松和监测骨丢失方面具有很大的潜力,为骨质疏松早期诊断、骨丢失监测和骨髓间充质干细胞衰老评估提供了一种新的代谢标志物。
Description
技术领域
本发明涉及疾病诊断领域,具体地说,涉及血清半乳糖凝集素-1作为骨代谢标志物在骨质疏松诊断中的应用。
背景技术
老年人的骨质流失,通常被称为老年骨质疏松症,随着年龄的增长而发生,导致骨骼脆弱,增加骨折的风险。它已成为世界范围内影响老年人口最严重的疾病之一。老年骨质疏松症的病理生理机制是目前研究的热点,旨在开发新的方法来诊断、预防和治疗老年患者的骨质疏松性骨丢失。
骨是一种坚硬的组织,由骨基质和骨细胞组成。到目前为止,已经发现了两种主要的骨细胞:成骨细胞(负责骨形成)和破骨细胞(负责骨吸收)。它们的作用是建立一种被称为“骨重塑”的骨更新过程,通过骨吸收和骨形成来维持骨骼稳态。在骨稳态中,破骨细胞移除的骨量一般与成骨细胞形成的骨量相等。然而,骨内稳态在衰老过程中被破坏。骨吸收增加和/或骨形成减少可导致骨丢失。几项关于髂骨活检的组织形态学研究显示骨形成的减少而不是骨吸收的增加似乎是老年骨质疏松症的主要病理原因。
骨形成取决于骨重塑过程中成骨细胞的数量和活性。成骨细胞是由存在于骨髓非造血腔室中的干细胞(称为骨髓间充质干细胞,BMSCs)分化而来的。骨髓间充质干细胞的成骨分化经历了前成骨细胞、成骨细胞、成熟成骨细胞,最终形成细胞外基质的沉积和矿化。骨小梁较皮质骨更容易发生骨丢失,因为它经历了更广泛的骨重塑。因此,骨小梁较多的骨骼如椎骨、股骨、胫骨,更容易出现骨质疏松症。
到目前为止,骨质疏松症的诊断主要依赖于背痛的主诉,影像学上的骨骼变化,以及股骨和腰椎处的骨密度(BMD)。然而,包括骨丢失在内的影像学改变通常是中晚期骨质疏松症的征象。因此,骨转换增加的生化标志物已被提出作为骨丢失严重程度的潜在指标。累积的数据支持代表骨转换的标志物与骨质疏松症进展相关。这些标志物正在作为发现骨丢失、诊断早期骨质疏松症和监测疾病进展的生物标志物进行研究。
半乳糖凝集素-1(Galectin-1,Gal-1)是β-半乳糖凝集素家族成员之一,广泛分布于多种组织,参与众多生理过程,包括神经干细胞的生长、造血谱系和肌肉分化,并被证明在调控恶性肿瘤进程方面具有重要作用,可促进肿瘤细胞的生长、侵袭、转移及血管生成,可能成为恶性肿瘤的早期诊断、生物治疗以及预后判断的重要标志和靶点。
而目前未见半乳糖凝集素-1在骨质疏松诊断中的应用。
发明内容
本发明的目的是针对现有技术中的不足,提供血清半乳糖凝集素-1作为骨代谢标志物在骨质疏松诊断中的应用。
第一方面,本发明提供了检测半乳糖凝集素-1蛋白或其编码基因表达水平的试剂在制备骨质疏松诊断试剂盒中的应用。
作为本发明的一个优选例,所述试剂盒用于骨质疏松的早期诊断。
第二方面,本发明提供了检测半乳糖凝集素-1蛋白或其编码基因表达水平的试剂在制备监测骨丢失进展的试剂盒中的应用。
作为本发明的一个优选例,所述监测骨丢失进展为监测骨体积分数、骨小梁数目和/或骨小梁分离度。
第三方面,本发明提供了检测半乳糖凝集素-1蛋白或其编码基因表达水平的试剂在制备评估骨髓间充质干细胞衰老程度的试剂盒中的应用。
针对以上各技术方案,作为其中的一个优选例,所述试剂盒用于检测血清、血浆、全血、骨髓间充质干细胞或骨髓基质细胞中半乳糖凝集素-1蛋白或其编码基因的表达水平。
作为其中的另一优选例,所述检测半乳糖凝集素-1蛋白或其编码基因表达水平的试剂是特异性扩增半乳糖凝集素-1蛋白的编码基因的引物;或特异性识别半乳糖凝集素-1蛋白的编码基因或其转录本的探针;或特异性结合半乳糖凝集素-1蛋白的结合分子;或特异性识别半乳糖凝集素-1蛋白的编码基因或其转录本的芯片。
更优选地,所述试剂盒还包括:核酸抽提试剂;聚合酶链反应试剂;蛋白免疫组化试剂;酶链免疫反应试剂;或使用说明书。
本发明优点在于:
老年性骨质疏松症与年龄相关的骨丢失是根据骨的影像学变化和骨密度(BMD)测量来诊断的。然而,影像学改变通常是中晚期骨质疏松症的征象。因此,生物标志物被提出作为骨丢失的指标。在本研究中,Galectin-1(Gal-1)在小鼠和临床受试者血清中表现出与年龄相关的下降。Gal-1在骨质疏松症中的作用目前尚未被研究。因此,我们的研究阐明了血清Gal-1水平与骨丢失的关系。
方法:采用6月龄和18月龄小鼠建立年龄相关性骨丢失动物模型,采用Micro-CT观察其骨密度和显微结构。Elisa法检测血清骨转换标志物和Gal-1水平。通过相关性分析说明血清Gal-1水平与骨丢失之间的关系。此外,采用免疫组化方法检测小鼠骨髓中Gal-1的表达水平。采用Elisa和western blot方法分别检测Gal-1在骨髓基质细胞(BMSC)、造血干细胞(HSC)和骨髓脂肪组织(BMAT)中的分泌能力和蛋白表达。流式细胞术检测骨髓中骨髓间充质干细胞的数量。最后,招募年龄相关性骨密度下降的男性志愿者,分析血清Gal-1与骨密度的关系。
结果:小鼠血清半乳糖凝集素-1(Gal-1)呈年龄相关性下降。血清Gal-1与小鼠股骨、胫骨和L1椎体的BV/TV呈正相关。与HSC和BMAT相比,骨髓间充质干细胞分泌更多Gal-1。老年小鼠骨髓中骨髓间充质干细胞的数量明显低于幼龄小鼠。老年小鼠骨髓间充质干细胞中Gal-1蛋白表达明显低于幼龄小鼠。此外,我们发现与年龄相关的骨密度下降的男性血清Gal-1水平下降。这些男性的骨密度与血清Gal-1水平呈正相关。
结论:年龄相关性骨量丢失与小鼠和受试者血清Gal-1水平下降有关。我们的研究表明,Gal-1在发现骨髓间充质干细胞衰老、诊断早期骨质疏松和监测骨丢失方面具有很大的潜力。
附图说明
图1:小鼠年龄相关的骨小梁丢失。股骨(A)、胫骨(C)和L1椎骨(E)取自6个月和18个月大的小鼠。应用Micro-CT对骨小梁显微结构进行评估。骨小梁参数包括BV/TV、Tb.N、Tb.Sp。根据显微ct扫描进行定量。股骨远端(B)、胫骨近端(D)和L1椎体(F)为代表性骨小梁三维重建图像。从6月龄和18月龄小鼠的外周血中制备血清。采用Elisa测定骨转换标志物CTX-I和PINP(G)的水平。数据以均数±标准差表示。*:p<0.05,**:p<0.01,***:p<0.001,18个月vs.6个月。
图2:C57BL/6小鼠与增龄相关的骨量丢失。股骨(A)、胫骨(C)和L1椎体(E)取自6个月和18个月大的C57BL/6小鼠。采用Micro-CT评价骨小梁体积分数(BV/TV)。显示股骨远端(B)、胫骨近端(D)、L1椎体(F)代表性的骨小梁三维重建图像。从6月龄和18月龄C57BL/6小鼠外周血制备血清。采用Elisa法定量测定骨转换标志物CTX-I和PINP(G)的水平,数据以均数±标准差表示。*:p<0.05,**:p<0.01,18月龄vs 6月龄。
图3:18月龄小鼠外周血血清和骨髓微环境中Gal-1水平与年龄相关的下降,以及骨小梁体积分数与Gal-1水平的相关性。从6月龄和18月龄小鼠的外周血中制备血清。Elisa测定Gal-1水平(A)。分析血清Gal-1水平与股骨(B)、胫骨(C)和L1椎体(D)的BV/TV的相关性。从6个月和18个月大的小鼠中制备股骨骨髓抽吸物。采用Elisa法定量检测Gal-1的表达(E),免疫组化法检测6月龄和18月龄小鼠股骨骨髓中Gal-1的表达(F),分析骨髓中Gal-1水平与股骨BV/TV的相关性。数据以均数±SD表示。*:p<0.05,***:p<0.001,18个月vs.6个月。
图4:6月龄和18月龄Balb/c小鼠外周血中细胞因子的分泌。从6月龄和18月龄Balb/c小鼠外周血制备血清。采用抗体阵列检测细胞因子TNFα(A)、bFGF(B)、IL-11(C)、IL-17(D)、CCL2(E)、CXCL1(F)和SDF-1α(G)的水平,数据以均数±标准差表示。
图5:18月龄C57BL/6小鼠外周血和骨髓微环境中Gal-1水平的年龄相关性以及C57BL/6小鼠小梁骨体积分数与Gal-1水平的相关性。从6月龄和18月龄C57BL/6小鼠外周血制备血清。采用Elisa法测定血清Gal-1水平(A),分析血清Gal-1水平与股骨(B)、胫骨(C)、L1椎体(D)BV/TV的相关性。取6月龄和18月龄C57BL/6小鼠制备股骨骨髓液。采用Elisa法定量(E),分析骨髓Gal-1水平与股骨BV/TV(F)的相关性。数据以均数±标准差表示。*:p<0.05,**:p<0.01,18月龄vs 6月龄。
图6:骨髓微环境中年龄相关的骨髓间充质干细胞数量减少。从6月龄小鼠股骨骨髓中提取骨髓间充质干细胞(BMSC)、造血干细胞(HSC)和BMAT,体外培养。Elisa检测上清液中Gal-1的分泌情况(A),流式细胞术检测6月龄和18月龄小鼠股骨骨髓中CD73和Sca1阳性细胞的百分比(B)。数据以平均值±SD表示。**:p<0.01,***:p<0.001。
图7:6月龄和18月龄小鼠骨髓间质干细胞、HSC和BMAT中Gal-1蛋白表达的比较。取6月龄和18月龄小鼠股骨骨髓,取BMSC、HSC和BMAT。western blot检测6月龄和18月龄小鼠股骨骨髓间质干细胞(A)、HSC(B)和BMAT(C)中Gal-1蛋白水平。GAPDH作为内参。
图8:6月龄和18月龄C57BL/6小鼠骨髓间充质干细胞(BMSC)、HSC和BMAT中Gal-1蛋白表达的比较。BMSC(A)、HSC(B)和BMAT(C)取自6月龄和18月龄C57BL/6小鼠的股骨骨髓液。western blot检测Gal-1蛋白水平。以GAPDH为内参。
图9:不同年龄男性外周血血清中骨密度和Gal-1水平的下降以及骨密度与血清Gal-1水平的相关性。DEXA用于测量不同年龄组男性股骨颈(A)、全髋关节(B)和L1-L4腰椎(C)的骨密度。采用酶联免疫吸附法(Elisa)测定这些男性外周血血清中Gal-1水平(D)。分析血清Gal-1水平与骨密度(E、F、G)的相关性。数据以均数±SD表示。*:p<0.05,**:p<0.01,***:p<0.001。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1
一、材料与方法
1.动物
雄性Balb/c小鼠(上海sipri-bk实验动物有限公司,中国,上海)饲养在无特异性病原体(SPF)条件下。所有动物手术均通过上海建工医院动物伦理委员会批准。
每组有15只动物。采用异氟醚吸入麻醉,颈椎脱位,对小鼠实施安乐死。准备右股骨、胫骨和L1椎体进行显微计算机断层扫描;左股骨用于骨髓间充质干细胞(BMSC)、造血干细胞(HSC)和BMAT分离。取外周血血清测定CTX-I和PINP(免疫诊断系统plc,Tyne andWear,UK)。
2.Micro-CT
采用如文献(Zhou Y,Yang Y,Liu Y,Chang H,Liu K,Zhang X,et al.Irp2Knockout Causes Osteoporosis by Inhibition of Bone Remodeling.Calcif TissueInt.2019;104:70-8.)描述的方法行Micro-CT(SkyScan 1172;Bruker-microCT,Kontich,比利时)检查,通过内部体积绘制软件重建三维图像,该软件可以从任意视角和方向绘制CT扫描的三维视图,并测量骨微观结构参数。主要的骨参数是BV/TV(骨体积分数)、Tb.N(骨小梁数目)和Tb.Sp(骨小梁分离度)。
3.抗体
采用绝对定量夹心抗体阵列(小鼠细胞因子阵列q系列),按照文献(Wang Y,Han X,Yang Y,Qiao H,Dai K,Fan Q,et al.Functional differences betweenAMPKα1andα2subunits in osteogenesis,osteoblast-associated induction ofosteoclastogenesis,and adipogenesis.Sci Rep.2016;6:32771-84.)的方法检测小鼠外周血血清细胞因子水平。用于检测的抗体是经生物素标记的,被制备成鸡尾酒试剂备用。荧光检测的微阵列,每一个芯片都是单独的,以便每个阵列可以用不同的样品孵育。用阻断缓冲液孵育后,阵列与外周血血清样本孵育。洗涤去除非特异性结合后,加入生物素化检测抗体的混合物处理阵列。洗涤后,应用链霉亲和素结合荧光试剂孵育(HiLyte fluorTM532来自Anaspec,Fremont,CA)。然后使用基于激光的扫描系统(GenePix 4200A,分子动力学,森尼韦尔,加州)检测荧光信号。
4.酶联免疫吸附试验(Elisa)
根据说明书,用Elisa试剂盒(R&D,Minneapolis,MN,USA)测定小鼠骨髓抽吸液和外周血血清以及男性外周血血清中的Gal-1水平。
5.BMSC、HSC和BMAT分离培养
根据文献(Tencerova M,Figeac F,Ditzel N,H,KamillaNielsen T,Kassem M.High-Fat Diet-Induced Obesity Promotes Expansion of BoneMarrow Adipose Tissue and Impairs Skeletal Stem Cell Functions in Mice.J BoneMiner Res.2018;33:1154-65.)的方法,分离BMSC、HSC和BMAT。骨髓悬浮在含有2%胎牛血清的冷PBS中,通过70μm过滤器过滤。滤过的骨髓细胞置于含有2%胎牛血清和0.1g/L酚红的PBS中,然后进行CD45、CD31、Ter119细胞的阴性选择,获得骨髓间充质干细胞。含基质细胞的阳性部分也保留作进一步分析。细胞体外培养并扩增至第3代。
采用冲洗骨髓和快速高速旋转的方法从长骨中分离BMAT。然后将含有BMAT和基质细胞的浑浊液用PBS重悬,使BMAT浮在上方。用红细胞裂解液溶解星状细胞颗粒以清除红细胞。
6.免疫蛋白印迹技术
按照文献(Fan Q,Tang T,Zhang X,Dai K.The role of CCAAT/enhancerbinding protein(C/EBP)-alpha in osteogenesis of C3H10T1/2cells induced byBMP-2.J Cell Mol Med.2009;13:2489-505.)的方法进行Western blot。细胞在含有50mMTris-HCl,pH 7.4,150mM NaCl,1%Noidet P-40和0.1%SDS并添加了蛋白酶抑制剂(10g/ml leupeptin,10g/ml pepstatin A和10g/ml抑肽酶)的缓冲液中冰浴30分钟。用SDS-PAGE分离蛋白,转移到PVDF膜上,用抗galectin-1(#5418,Cell Signaling Technology,Danvers,MA,USA)和抗GAPDH(#2118,CST)检测。使用增强化学发光系统(GE Healthcare,Piscataway,NJ,USA)对蛋白质进行荧光检测。
7.免疫组织化学方法
参考文献(Li X,Zhou Z,Zhang Y,Yang H.IL-6Contributes to the DefectiveOsteogenesis of Bone Marrow Stromal Cells from the Vertebral Body of theGlucocorticoid-Induced Osteoporotic Mouse.PLoS One.2016;11:e0154677.)的方法,将股骨固定在10%的福尔马林中,脱钙,石蜡包埋。每5μm连续切片。载玻片与抗小鼠半乳糖凝集素-1(#13888,CST)的一抗在4℃孵育过夜。免疫组化染色采用辣根过氧化物酶-链霉亲和素检测系统(Dako)检测免疫活性,然后用苏木精(Dako)进行反染。
8.流式细胞技术
参考文献(Wu X,Cao L,Li F,Ma C,Liu G,Wang Q.Interleukin-6 fromsubchondral bone mesenchymal stem cells contributes to the pathologicalphenotypes of experimental osteoarthritis.Am J Transl Res.2018;10:1143-54.)的方法,收集股骨和胫骨的骨髓细胞并集中在一起。红细胞裂解后,骨髓细胞离心,混悬液重悬,4%多聚甲醛固定。用0.1%Triton X-100渗透细胞,然后用封闭缓冲液(含3%胎牛血清和0.1%叠氮化钠的PBS)冰敷30分钟。然后将细胞与anti-CD73(12-0731-83,ThermoFisherscientific)、anti-Sca1(11-5981-85,ThermoFisher scientific)或同型对照在暗室37℃孵育1小时,再用含0.1%BSA的PBS洗2次。探针使用BD Calibur流式细胞仪和CellQuest软件(Becton Dickinson)进行分析。
9.临床试验对象
参考文献(Jiang Y,Zhang Y,Jin M,Gu Z,Pei Y,Meng P.Aged-Related Changesin Body Composition and Association between Body Composition with Bone MassDensity by Body Mass Index in Chinese Han Men over 50-year-old.PLoS One.2015;10:e0130400.)的方法,本研究招募并评估了82名在我院进行常规体检和DEXA检查的中国男性患者。排除标准为代谢性骨病史,如慢性肝肾衰竭、甲状腺功能亢进和类风湿性关节炎;有影响体重或身体组成的疾病史,如甲状腺机能亢进、甲状腺功能减退;存在使人衰弱的重大疾病;主要心血管事件;所有受试者均未出现原发性或继发性低性腺激素水平,或在前3个月内使用过影响骨密度、体重及身体成分的药物,如甲状腺激素、糖皮质激素、双磷酸盐、减肥药物等。将82名男性分为3组:年龄30-39岁(第1组,n=23)、45-54岁(第2组,n=32)、65-74岁(第3组,n=27)。
本研究经上海市建工医院(中华人民共和国,上海)伦理委员会批准。调查人员遵守所有适用的规章和法律要求以及1975年以来的《赫尔辛基宣言》(1983年订正)。在纳入研究之前,每位受试者均提供了书面知情同意,且在未给予适当书面知情同意的情况下,受试者均未参与任何与研究相关的活动。在整个研究过程中,受试者被严格保密。
10.骨密度测定
通过DEXA(Lunar iDXA,General Electric Company,Fairfield,USA)对股骨颈、全髋关节和腰椎(L1-L4)进行扫描来评估所有受试者的骨密度。用骨面积和骨矿物质含量评分计算骨密度(g/cm2)。所有的扫描结果都是由同一名经验丰富的操作员采集和分析的,并遵循制造商提供的指导方针。
11.统计学方法
采用t检验计算两样本间的统计学差异。方差分析在SPSS 16.0软件中进行多重比较。采用Spearman相关分析计算血清Gal-1水平与骨小梁体积分数的相关性。p<0.05为差异具有统计学意义。除非另有说明,所有数据均以平均值±SD表示。
2.结果
2.1小鼠与年龄相关的小梁骨丢失
小鼠骨量峰值出现在5~6月龄。因此,本研究使用6个月和18个月大的Balb/c小鼠来研究年龄相关的骨小梁丢失。收集股骨、胫骨和L1椎体。Micro-CT检测骨小梁的显微结构。
与6个月大的小鼠相比,18个月大的小鼠股骨远端BV/TV下降了25%,Tb.N下降了27%,Tb.Sp增加了24%(图1A)。与6个月大的小鼠相比,18个月大的小鼠胫骨近端BV/TV下降29%,Tb.N下降24%,Tb.Sp增加48%(图1C)。与6月龄小鼠相比,18月龄小鼠L1椎骨的BV/TV下降了25%,Tb.N下降了20%,Tb.Sp增加22%(图1E)。骨小梁三维重建图像也显示相同的情形(图1B、1D、1F)。
除Micro-CT扫描外,采用Elisa法检测6月龄和18月龄小鼠外周血中骨形成和骨吸收标志物,以确定骨转换状态。6个月和18个月的小鼠骨吸收的标志——c端I型胶原(CTX-I)没有显著变化(图1G)。然而,与6个月大的小鼠相比,在18个月大的小鼠血清中观察到I型前胶原前肽(PINP)显著减少(超过36%),PINP是骨形成的标志。
为了消除小鼠品系偏差,除了Balb/c小鼠外,我们还研究了6月龄和18月龄C57BL/6小鼠的血清骨小梁显微结构和骨转换标志物。ct机和Elisa结果显示了与Balb/c小鼠类似的情形,即:与6个月大的小鼠相比,18个月大的C57BL/6小鼠有显著的股骨(图2A和2B)、胫骨(图2C和2D)和L1椎骨(图2E和2F)骨小梁丢失以及血清PINP水平的降低(图2G),虽然变化范围在Balb/c和C57BL/6小鼠之间是不同的。
2.2小鼠年龄相关性骨小梁丢失与血清Gal-1水平下降的相关性
制备6月龄和18月龄Balb/c小鼠外周血血清。抗体阵列被用来量化几种重要的细胞因子的水平。与6个月大的小鼠相比,18个月大的小鼠血清中Gal-1的分泌显著减少(图3A)。6月龄和18月龄小鼠TNFα(图4A)、bFGF(图4B)、IL-11(图4C)、IL-17(图4D)、CCL2(图4E)、CXCL1(图4F)和SDF-1α(图4G)的分泌水平无显著差异。同样,与6个月大的C57BL/6小鼠相比,18个月大的C57BL/6小鼠的血清Gal-1水平也出现了年龄相关的下降(图5A)。
观察到小鼠年龄相关的骨小梁丢失和血清Gal-1水平下降,我们想知道它们是否相关。为了回答这个问题,我们探讨了血清Gal-1水平与股骨、胫骨和L1椎体的骨小梁体积分数(BV/TV)的相关性。结果显示,血清Gal-1水平与股骨BV/TV(r=0.638,p<0.001)(图3B和表1)、胫骨BV/TV(r=0.511,p<0.01)(图3C和表1)和L1椎体BV/TV(r=0.652,p<0.001)呈正相关(图3D和表1)。在C57BL/6小鼠中,血清Gal-1水平与股骨BV/TV(r=0.6814,p<0.01)(图5B)、胫骨BV/TV(r=0.6619,p<0.01)(图5C)和L1椎体BV/TV(r=0.6312,p<0.01)呈正相关(图5D)。
2.3小鼠骨髓微环境中Gal-1水平与年龄相关的下降
分别从6月龄和18月龄Balb/c和C57BL/6小鼠股骨中制备骨髓抽吸物。Elisa法测定Gal-1水平。与6个月大的小鼠相比,观察到18个月大的小鼠股骨骨髓抽吸液中Gal-1水平显著降低(图3E和5E)。免疫组化结果也显示,与6月龄小鼠相比,18月龄小鼠股骨骨髓中Gal-1表达下调(图3F)。我们探讨了骨髓抽吸物中Gal-1水平与股骨BV/TV的相关性。结果显示,骨髓抽吸物中的Gal-1水平与Balb/c和C57BL/6小鼠股骨BV/TV呈正相关(图3G和表1)(图5F)。
2.4年龄相关性降低骨髓间充质干细胞数量,下调骨髓间充质干细胞Gal-1表达
从6月龄小鼠骨髓中提取骨髓间充质干细胞(BMSC)、造血干细胞(HSC)和骨髓脂肪组织(BMAT)进行体外培养。Elisa检测上清中Gal-1的分泌情况。骨髓间质干细胞分泌的Gal-1比培养的HSC高2倍,比培养的BMAT高18倍(图6A)。流式细胞术检测6月龄和18月龄小鼠骨髓中CD73和Sca1阳性细胞的百分率。结果显示,18月龄小鼠骨髓中CD73和Sca1阳性细胞的百分比明显低于6月龄小鼠(图6B)。
从6月龄和18月龄Balb/c和C57BL/6小鼠骨髓中提取BMSC、HSC和BMAT。Westernblot检测Gal-1蛋白在不同类型细胞中的表达。与6月龄小鼠相比,18月龄小鼠骨髓间质干细胞(图7A和8A)和HSC(图7B和8B)中Gal-1蛋白表达显著下调。6月龄和18月龄小鼠的BMAT中Gal-1蛋白表达水平无差异(图7C和8C)。
2.5不同年龄男性骨密度与血清Gal-1水平的关系
在该研究中,招募不同年龄的男性志愿者。表2总结了所有登记男性的基本特征。分为3组:30-39岁(组1)、45-54岁(组2)、65-74岁(组3)。在三个年龄组中,身体质量指数(BMI)没有显著区别(表2)。双能x线骨密度仪(DEXA)用于测量股骨颈(FN)、全髋关节(TH)和L1-L4腰椎的骨密度。在FN BMD,与组1相比,组2和组3显著减少(图9A和表2)。在TH BMD,与组1比较,组3有明显降低(图9B和表2)。在L1-4 BMD,与组1比较,组2和组3有明显降低(图9C和表2)。采用Elisa法检测外周血血清Gal-1水平。结果显示,与组1相比,组2、组3血清Gal-1水平明显降低(图9D、表2),且组3血清Gal-1水平较组2降低,也具有统计学意义(图9D、表2)。
在观察到年龄相关的男性骨密度下降和血清Gal-1水平下降后,我们想到了他们是否相关。为了回答这个问题,我们探讨了血清Gal-1水平与FN、TH和L1-4腰椎骨密度的相关性。血清Gal-1水平不同程度与FN BMD(r=0.4552,p<0.001)(图9E和表3)、TH BMD(r=0.3269,p<0.01)(图9F和表3)和L1-4 BMD(r=0.4654,p<0.001)呈正相关(图9G和表3)。
3.结论
在小鼠和男性中,年龄相关的骨小梁丢失与血清Gal-1水平下降相关。年龄相关性的骨髓间充质干细胞数量的减少以及骨髓间充质干细胞和HSC对Gal-1表达的抑制是导致小鼠骨髓和血清中Gal-1水平下降的原因之一。本研究中小鼠实验和临床样本评估的数据表明,Gal-1在发现骨髓间充质干细胞衰老、诊断早期骨质疏松症和监测疾病进展方面具有很大的潜力。
4.参与的伦理批准和同意
所有动物手术均通过上海建工医院动物伦理委员会批准。所有涉及动物的程序都是按照《入境指南》执行的。
本研究经上海市建工医院(中华人民共和国上海)伦理委员会批准。调查人员遵守了所有适用的规章和法律要求以及1975年以来的《赫尔辛基宣言》(1983年订正)。在纳入研究之前,每位受试者均提供了书面知情同意,且在未给予适当书面知情同意的情况下,受试者均未参与任何与研究相关的活动。在整个研究过程中,受试者被严格保密。
表1:血清和骨髓Gal-1水平与股骨、胫骨和L1椎骨BV/TV的相关性
表2:不同年龄组男性人口统计学数据、血清Gal-1水平和股骨颈、全髋关节、腰椎骨密度的比较
注:所有数据均以平均值±标准差表示。*:p<0.05,***:p<0.001,组2vs组1。##:p<0.01,###:p<0.001,组3vs组1。$$:p<0.01,组3vs组2。
BMI:体重指数;BMD:骨密度;FN:股骨颈;TH:全髋;L1-4腰椎。
表3:血清Gal-1水平与男性股骨颈、全髋、L1-L4骨密度的相关性
注:BMI:体重指数;BMD:骨密度;FN:股骨颈;TH:全髋;L1-4腰椎。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (8)
1.检测半乳糖凝集素-1蛋白或其编码基因表达水平的试剂在制备骨质疏松诊断试剂盒中的应用。
2.根据权利要求1所述的应用,其特征在于,所述试剂盒用于骨质疏松的早期诊断。
3.检测半乳糖凝集素-1蛋白或其编码基因表达水平的试剂在制备监测骨丢失进展的试剂盒中的应用。
4.根据权利要求3所述的应用,其特征在于,所述监测骨丢失进展为监测骨体积分数、骨小梁数目和/或骨小梁分离度。
5.检测半乳糖凝集素-1蛋白或其编码基因表达水平的试剂在制备评估骨髓间充质干细胞衰老程度的试剂盒中的应用。
6.根据权利要求1-5任一所述的应用,其特征在于,所述试剂盒用于检测血清、血浆、全血、骨髓间充质干细胞或骨髓基质细胞中半乳糖凝集素-1蛋白或其编码基因的表达水平。
7.根据权利要求1-5任一所述的应用,其特征在于,所述检测半乳糖凝集素-1蛋白或其编码基因表达水平的试剂是特异性扩增半乳糖凝集素-1蛋白的编码基因的引物;或特异性识别半乳糖凝集素-1蛋白的编码基因或其转录本的探针;或特异性结合半乳糖凝集素-1蛋白的结合分子;或特异性识别半乳糖凝集素-1蛋白的编码基因或其转录本的芯片。
8.根据权利要求7所述的应用,其特征在于,所述试剂盒还包括:核酸抽提试剂;聚合酶链反应试剂;蛋白免疫组化试剂;酶链免疫反应试剂;或使用说明书。
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