CN113514643A - 剪接相关蛋白130用于特发性肺间质纤维化诊断和疾病严重程度评估 - Google Patents
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Abstract
本发明涉及剪接体相关蛋白130(SAP130)用于特发性肺间质纤维化诊断和疾病严重程度评估。本发明经临床病例研究发现剪接体相关蛋白130可用于特发性肺间质纤维化诊断,并可用于疾病严重程度评估。该方法直接抽取患者外周血进行SAP130检测,采用ELISA方法,检测简便,检测时间短。诊断效能高,诊断敏感性高。本发明为特发性肺纤维化的诊断和病情评估提供了一种新的手段。
Description
技术领域
本发明涉及生物医药领域,尤其涉及剪接体相关蛋白130用于特发性肺间质纤维化诊断和疾病严重程度评估。
背景技术
特发性肺纤维化(Idiopathic pulmonary fibrosis,IPF)是常见的肺间质疾病,以肺纤维化为特点,患病率约(2~29)/10万,中位生存期仅3年左右。IPF在男性中更为常见,并且患病率随着年龄的增长而明显增加。全球IPF患者死亡率呈现上升趋势。IPF的主要症状表现为进行性呼吸困难,最终导致呼吸衰竭或死亡,病理特征主要为异常的细胞外基质沉积以及肺组织结构重塑异常。目前越来越多的研究认为肺纤维化是持续性肺损伤和异常修复的结果,即当肺上皮细胞受到持续性损伤时,肺组织异常修复,肺间质细胞大量增殖,产生过多的胶原以及细胞外基质,正常肺泡结构丧失,被异常增殖的纤维组织及细支气管化的囊性气腔结构所取代,造成不可逆的肺纤维化。
剪接体相关蛋白130(Spliceosome-associated protein,SPAl30)作为巨噬细胞诱导型C型凝集素(Macrophage-inducible C-type lectin,Mincle)的一种结合蛋白,是U2snRNP的一个组件,固定存在于活细胞的细胞核中。当细胞损伤或坏死时SAP130可以从细胞核中释放,从而与Mincle受体结合,从而激活免疫反应。Yamasaki S等建立了表达Mincle和FcRγ的报告细胞系,当Mincle被配体激活时,将激活NFAT并导致绿色荧光蛋白的表达,绿色荧光蛋白阳性活细胞的数量随坏死细胞的数量而增加,提示Mincle可以作为坏死细胞的传感器。从死细胞裂解物中纯化出SAP-130作为选择性结合Mincle-Ig融合蛋白的候选蛋白。SAP130升高见于酒精性肝损伤、胰腺导管腺癌、自身免疫性肝炎等疾病。原代肝细胞接受乙醇刺激后24小时,细胞培养基上清液SAP130表达增加,系乙醇致肝细胞内质网应激、线粒体功能障碍而发生细胞凋亡或坏死,释放SAP130。免疫组化研究显示胰腺导管细胞癌肿瘤组织中导管细胞癌细胞释放SAP130蛋白增加,并可激活Mincle。USSC RNA测序显示胰腺肿瘤组织高表达SAP130患者较低表达患者的生存率低。细胞水平研究显示人转移胰腺癌细胞经与吉西他滨处理后SAP130较未处理的肿瘤细胞的SAP130表达量下降。加用RIRK抑制剂Nec-1s可抑制胰腺肿瘤细胞产生SAP130,提示胰腺肿瘤组织SAP130升高与肿瘤细胞细胞程序性坏死有关。
而目前尚无SAP130在IPF疾病表达的相关报道。
发明内容
本发明基于临床研究发现IPF患者血清SAP130高于健康人,并且血SAP130水平与IPF疾病严重程度之间存在正相关性,提示SAP130可以反映IPF疾病严重度。基于此完成了本发明。
本发明首先提供了检测剪接体相关蛋白130或其基因表达量的试剂在制备特发性肺纤维化诊断试剂中的用途。
本发明又提供了剪接体相关蛋白130作为生物标志物在制备特发性肺纤维化诊断试剂中的用途。
本发明又提供了检测剪接体相关蛋白130或其基因表达量的试剂在制备特发性肺纤维化严重程度评估试剂中的用途。
本发明又提供了剪接体相关蛋白130作为生物标志物在制备特发性肺纤维化严重程度评估试剂中的用途。
作为一个优选的实施方式,检测样品为血清。
作为另一优选的实施方式,剪接体相关蛋白130的临界值为643.87pg/ml。
作为另一优选的实施方式,所述试剂进一步被包装成试剂盒。
本发明的有益效果在于:
本研究临床发现IPF患者血清SAP130高于健康人,并且血SAP130水平与IPF疾病严重程度之间存在正相关性,提示SAP130可以反映IPF疾病严重度。IPF患者肺组织免疫组化显示SAP130高表达。第一秒用力呼气容积(Forced expiratory volume in 1second,FEV1)是反映肺通气功能的重要指标。一氧化碳弥散量(Carbon monoxide diffusing capacity,DLCO)是确定肺功能损害以及监测和量化IPF疾病进展的较为客观指标。IPF中的SAP130水平与DLCO和FEV1呈负相关。稳定期患者的血SAP130水平与HRCT的纤维化程度呈正相关。此外,在IPF急性加重患者(AE-IPF)中,不仅纤维化程度而且高分辨CT上磨玻璃影与血清SAP130水平有关。新发广泛的GGO区域提示广泛的肺泡上皮细胞损伤,细胞炎症因子和趋化因子表达增加。反复或持续的上皮损伤是导致肺纤维化的促炎和纤维化因子的诱发因素。IPF患者血清SAP130升高与肺上皮细胞损伤后SAP130从细胞核中释放,自细胞逸出至细胞间隙再入血。SAP130水平可反映IPF患者的病情严重程度,是肺纤维化的新型损伤相关模式,可作为临床上IPF诊断、疾病严重程度的生物标志物。
本发明属首次发现SAP130可用于特发性肺间质纤维化诊断,并可用于疾病严重程度评估。该方法直接抽取患者外周血进行SAP130检测,采用ELISA方法,简便快速。诊断效能高,诊断敏感性高。
附图说明
图1:(A)ELISA检测IPF患者和健康志愿者血清SAP水平。(B)ELISA检测稳定期IPF患者和AE-IPF患者血清SAP水平。
图2:ROC曲线显示血清SAP130水平诊断IPF(vs.健康志愿者)的特异性和敏感性。
图3:IPF稳定期患者血SAP130与临床参数的相关性。(A)FEV1(%predicted);(B)DLCO(%predicted);(C)纤维化(%)。
图4:AE-IPF患者中SAP130与临床参数的相关性。(A)纤维化(%);(B)GGO(%)。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
实施例1
1、方法
为研究SAP130与IPF的临床相关性,首先使用ELISA方法检测比较了83位IPF患者和38位健康对照的SAP130水平。IPF患者和对照的人血清样本和肺组织切片的来自瑞金医院。所有实验均经医院伦理委员会审批同意。入组时间为2017年1月至2019年12月。免疫组化和免疫荧光所用肺组织蜡块系接受肺移植的IPF患者手术切除下来原肺脏器。对照组肺组织来自某医院因肺部结节而行手术的患者,术后病理确诊为良性结节。所用肺组织尽可能远离病灶。IPF患者血清来国内六家医院。外周血血清检测SAP130健康对照组系按年龄和性别相匹配,从某医院健康检查中心招募。这些健康受试者在低剂量胸部计算机断层扫描中没有发现肺部疾病的证据。分析IPF患者血清SAP与临床指标参数的相关性。所有患者均经过临床,影像学和生理评估。初次就诊时收集了有关IPF稳定期患者的一般特征,六分钟步行测试(6MWT),肺功能检查和胸部高分辨率CT的数据。计量资料数值结果采用平均数±标准差表示。两个样本间的差异使用双侧student t检验(two-tailed Student’t-test)或者双因素方差分析检验(two-way ANOVA)。使用ROC曲线分析血清SAP130水平用于区分IPF患者和健康对照组以及稳定期IPF和AE-IPF患者的临床效能。Spearman分析评估血清SAP130水平与各种临床和实验室参数之间的相关性。实验结果用GraphPad Prism 7软件和SPSS 21.0软件分析。*P<0.05,**P<0.01,***P<0.001,NS表示无统计学差异。当P<0.05时,实验结果的差异有统计学意义。
ELISA方法如下:使用前,将ELISA检测试剂和IPF患者血清样品置于室温。所有血清样品均一式两份进行测定。在预包被SAP130检测抗体的96孔板每孔添加100μL ELISA检测标准液和样品,用薄膜覆盖防止蒸发。在37℃下孵育2小时。吸出每个孔并洗涤(此时薄膜是覆盖的,丢弃标准液,保留样品),重复该过程,PBST(phosphate buffered solution)(250μL)共进行3次洗涤。最后一次洗涤后,通过抽吸或倾析除去PBST。翻转板并将其用干净的纸巾吸干。向每个孔中添加100μL生物素结合物(1×),37℃下孵育1小时。弃混合液并洗涤3次,重复共进行3次PBST洗涤。每孔加入100μL的链霉亲和素-过氧化物酶(HRP)(1×),37℃孵育1小时。吸出并PBST五次洗涤。每孔加100μL四甲基联苯胺(TMB)溶液。37℃孵15-20分钟。当含有最高浓度标准品的前四个孔出现明显的蓝色时,每孔加入50μL TMB反应终止液。450nm的酶标仪在5分钟内测定每个孔的吸光度。
2、结果
2.1不同组别血清SAP130水平比较
IPF患者的血清SAP130水平显著高于对照组(824.2±29.84vs.413.8±19.77pg/ml,P<0.0001)。AE-IPF患者的血清SAP130水平显著高于稳定期IPF患者(763.9±37.16vs.911.1±46.04pg/ml,P=0.0144)(图1)。
2.2血清SAP130水平诊断IPF的特异性和敏感性
相对于健康对照组,SAP130蛋白诊断IPF的ROC曲线下面积为0.944(95%CI,0.81-0.997),最佳临界值(643.87pg/ml),敏感性和特异性分别为92.1%和69.9%(图2)。
2.3稳定期IPF患者中SAP130与临床参数的相关性
Spearman的分析显示,稳定期IPF患者的血清SAP130水平与HRCT的纤维化水平和KL-6水平呈正相关(r=0.4164,P=0.0029;r=0.4564,P=0.0010),而与FEV1(%predicted)和DLCO(%predicted)呈负相关(r=-0.3562,P=0.0120;r=-0.5550,P<0.0001)(图3)。
2.4 AE-IPF患者中SAP130与临床参数的相关性
AE-IPF患者的血清SAP130与GGO和纤维化的总体程度呈正相关(r=0.4697,P=0.0051;r=0.3735,P=0.0296;r=0.5470,P=0.0008)(图4)。
上述实施例仅例示性说明本发明的原理及其功效,而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下,对上述实施例进行修饰或改变。因此,举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变,仍应由本发明的权利要求所涵盖。
Claims (7)
1.检测剪接体相关蛋白130或其基因表达量的试剂在制备特发性肺纤维化诊断试剂中的用途。
2.剪接体相关蛋白130作为生物标志物在制备特发性肺纤维化诊断试剂中的用途。
3.检测剪接体相关蛋白130或其基因表达量的试剂在制备特发性肺纤维化严重程度评估试剂中的用途。
4.剪接体相关蛋白130作为生物标志物在制备特发性肺纤维化严重程度评估试剂中的用途。
5.根据权利要求1-4任一权利要求所述的用途,其特征在于,检测样品为血清。
6.根据权利要求1-4任一权利要求所述的用途,其特征在于,剪接体相关蛋白130的临界值为643.87 pg/ml。
7.根据权利要求1-4任一权利要求所述的用途,其特征在于,所述试剂进一步被包装成试剂盒。
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