CN116656800A - 狼疮性肾炎标志物及用途 - Google Patents
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Abstract
本发明涉及狼疮性肾炎诊断治疗领域,尤其公开了一种狼疮性肾炎标志物及用途,本发明提供了定量检测标志物的试剂在制备预后评估、诊断或监测狼疮性肾炎试剂盒中的应用,所述标志物为cDC2,cDC2的标记基因为CD1c,本发明通过试验发现,与健康对照组相比,LN肾脏中T细胞、B细胞和髓样细胞显著富集。我们进一步分析了每种细胞类型与临床参数之间的相关性:Th17细胞、2型常规树突状细胞(cDC2)和iPT细胞与24h‑Upro呈正相关,与eGFR呈负相关,表明它们与LN疾病严重程度相关,即Th17、cDC2和iPT细胞比例越高,肾脏病变越严重。同时,在这些细胞类型中,cDC2与24h‑Upro和eGFR的相关性最强,由上述数据表明,Th17、cDC2和iPT可作为狼疮性肾炎的标志物,用于该疾病的预后评估、诊断或监测。
Description
技术领域
本发明涉及狼疮性肾炎诊断治疗领域,尤其涉及狼疮性肾炎标志物及用途。
背景技术
狼疮性肾炎(LN)是系统性红斑狼疮(SLE)最严重的表现之一,影响50~80%的SLE患者。尽管采用了先进的免疫抑制疗法,但仍有高达60%的LN患者无法获得完全缓解,而这些患者中有10~20%在10年内发展为终末期肾病(ESKD)。即使有精心设计的临床试验,也难以实现新的治疗性策略。这些不令人满意的情况增加了进一步研究疾病进展和个体异质性背后的致病机制的需要。
目前关于LN发病机制的知识表明,该疾病涉及多种细胞类型以及免疫和非免疫机制。B细胞可通过分泌直接针对结构细胞的自身抗体诱导肾损伤,而细胞毒性CD8+T细胞和CD4+T辅助(Th)细胞通过直接细胞毒性或促进B细胞分化和活化来驱动肾脏炎症。然而,小鼠研究表明,尽管有IC沉积,但Fcγ受体的缺乏或树突状细胞(DC)耗竭会消除LN肾脏中的T细胞活化和白细胞聚集,这意味着先天免疫细胞在人类疾病的免疫发病机制中的关键作用尚未明确。此外,肾结构细胞诸如内皮细胞、足细胞和肾小管上皮细胞被认为不仅是被动的受害者,而且是局部炎症的积极参与者。它们可在炎症状态期间通过免疫原性基因表达和细胞因子产生重塑肾脏微环境。尽管有多种细胞类型被认为与LN有关,并且它们复杂的细胞相互作用的结果与肾损伤程度密切相关,并可能影响LN患者的治疗结果,但它们的确切表型和在疾病进展中的作用仍不清楚。因此,对LN肾脏进行全面、深入的细胞分析,以鉴定出疾病相关联细胞类型,将有助于更好地理解致病机制,并为治疗性决策提供更精确的患者分层。
发明内容
本发明主要的目的是解决背景技术中存在的问题,为实现该目的,本发明提供以下技术方案:
定量检测标志物的试剂在制备用于预后评估、诊断或监测狼疮性肾炎试剂盒中的应用,所述标志物为cDC2(2型常规树突状细胞),所述cDC2的标记基因为CD1c。
进一步的,所述标志物还包括iPT(受损的近端肾小管上皮细胞)和/或Th1、Th17细胞。
优选的,所述Th1细胞的标记基因为IFNG。
优选的,所述Th17细胞的标记基因为IL17A。
优选的,所述iPT细胞的标记基因为SOX9和/或VCAM1。
在各个实施方案中,本文所述的用于预后评估、诊断或监测狼疮性肾炎试剂盒包括测定本发明所述的一种或多种标志物水平的试剂,例如对照、标准品和/或监测试剂等。在具体的一个实施例中,试剂盒具有实体形态,例如试剂盒可以是具有一个或多个空间的容器,该一个或多个空间用于容纳上述对照、标准品、检测试剂的材料或装置。
进一步的,本发明还提供了一种用于预后评估、诊断或监测狼疮性肾炎的方法为,测定在受试者的样品中所述标志物至少一种的数量,其中所述数量与所述受试者中狼疮性肾炎的严重程度呈正相关。
作为一种实施例,所述方法为当在测试样品中标志物的数量大于在来自相同受试者的早期样品中的数量时,鉴定出在受试者疾病的恶化,当在测试样品中标志物数量低于在早期样品中的数量时,鉴定出在受试者中疾病的改善。
进一步的,本发明还提供了治疗狼疮性肾炎的药物筛选方法,所述方法为在狼疮性肾炎患者服用药物前后,测试同一患者样品中权利要求1-5任一所述标志物的数量,当样品中标志物的数量低于服药前的数量,鉴定出在受试者中疾病的改善,药物有效。
进一步的,所述药物为免疫抑制剂和/或糖皮质激素。
进一步的,所述样品为肾活检穿刺获得的肾皮质和/或髓质。
本发明对40例LN患者的肾活检样本进行了单细胞RNA测序,其中7例患者与外周血样本配对,还有6例健康肾脏来自器官供体。我们观察到,与健康对照组相比,LN肾脏中T细胞、B细胞和髓样细胞显著富集。我们进一步分析了每种细胞类型与临床参数之间的相关性:Th17(cT03_CD4_IL17A)细胞、2型常规树突状细胞(cDC2)(cM02_cDC2_CD1C)和iPT细胞(cEpi04_iPT_SOX9)与24小时尿蛋白(24h-Upro)呈正相关,与估计肾小球滤过率(eGFR)呈负相关(图1f),表明它们与LN疾病严重程度相关,即Th17、cDC2和iPT细胞比例越高,肾脏病变越严重。同时,在这些细胞类型中,cDC2与24h-Upro和eGFR的相关性最强。由上述数据表明,Th17、cDC2和iPT可作为狼疮性肾炎的标志物,用于该疾病的预后评估、诊断或监测。
附图说明
图1:HC和LN患者肾脏的研究设计、注释和细胞组成图
a、用于处理肾活检和血样的研究和管道的设计。PBMC,外周血单核细胞;
b、来自HCs和LN患者肾活检样本的268,942个细胞的UMAP,其中51个免疫和肾结构细胞亚簇。ABC,年龄相关联B细胞;cDC,常规树突状细胞;pDC,浆细胞样树突状细胞;Neut,嗜中性粒细胞;Mono,单核细胞;Macro,巨噬细胞;VR,直小血管;GE,肾小球内皮细胞;VE,小静脉内皮;LE,淋巴管内皮;Podo,足细胞;iPodo,受损的足细胞;PT,近端肾小管;iPT,近端肾小管损伤;LOH,henle的环;DCT,远侧肾曲小管;PC,主细胞;ICA,A型闰细胞;ICB,B型闰细胞;Fib,成纤维细胞;aFib,活化成纤维细胞;Mesa,系膜细胞;Pe,周细胞;vSMC,血管平滑肌细胞;
c、显示HC和LN患者肾脏中免疫和肾结构细胞比例的条形图。HC,健康对照组;LN,狼疮性肾炎;
d、显示HC(n = 6)和LN患者(n = 40)肾脏中B细胞、T细胞、髓样细胞、内皮细胞、上皮细胞和间充质细胞的比例(相对于细胞总数)的箱线图。配对双侧Wilcoxon检验。*P<0.05,**P<0.01,***P<0.001,ns,不显著;
e、显示III/III+V LN类(n = 11)和IV/IV+V LN类(n =24)中B细胞、T细胞、髓样细胞、内皮细胞、上皮细胞和间充质细胞比例(相对于细胞总数)的箱线图。配对双侧Wilcoxon检验。*P<0.05,ns,不显著;
f、显示LN患者中B、T、髓样、内皮、上皮和间充质亚簇的比例(相对于对应谱系中细胞总数)与24UPO和eGFR之间的Pearson相关性的点图。
图2:cDC2亚簇的表征图
a、来源于HC和LN患者的肾活检样本的cDC2的UMAP,有4个亚簇;
b、显示标记基因表达的cDC2的UMAP;
c、显示LN患者中每个cDC2亚簇的比例(相对于髓样细胞总数)与24Upro和eGFR之间的Pearson相关性的点图;
d、显示了C0_DC3比例(相对于髓样细胞总数)与24UPO和eGFR之间的Pearson相关性的散点图;
e、LN肾脏中DC3的门控策略:DC3被定义为活的、单一的、LIN(CD3-CD19-CD56-)CD88-HLA-DR+CD11C+CD1C+CD163+细胞;
f、抗CD11c和CD163的LN患者肾活检切片的代表性mIHC染色示例,显示肾脏中的DC3。箭头表示特定的细胞类型。原始放大倍数,20倍;比例尺,50 μm。
图3:DC3对LN患者治疗反应的预测值效果图
a、显示完全缓解(CR,n = 11)和非完全缓解(NCR,n = 8)LN患者肾脏中DC3、Th1和Th17细胞比例的箱线图。非配对双侧Wilcoxon检验。CR,完全缓解;NCR,非完全缓解;
b、显示独立队列中CR(n = 30)和NCR(n = 30)LN患者肾脏中DC3、Th1和Th17细胞数目的箱线图。非配对双侧Wilcoxon检验;
c、用抗CD11c和CD163对肾活检切片进行mIHC染色的代表性示例显示了来自独立队列的有CR和NCR的LN患者中的DC3。原始放大倍数,20倍;比例尺,50 μm;
d、棒棒糖图显示了DC3计数、Th1和Th17细胞计数、人口统计学、临床和病理学特征在伴有CR和NCR的LN患者之间的单变量分析;
e、DC3数目、24hUpro、WBC、eGFR和肾小管坏死的单变量逻辑回归模型的ROC曲线。WBC,白细胞计数;
f、棒棒糖图显示LN患者CR和NCR之间的多变量分析。
图4来源于HC和LN患者肾活检样本的细胞的标记基因表达和分布图
a、显示肾脏中主要细胞类型的标准标记表达的UMAP;
b、由样本来源预测的总共51个免疫和肾结构细胞亚簇的UMAP。HC,健康对照组;LN,狼疮性肾炎。
图5 51个免疫和肾结构细胞亚簇的典型基因表达点图。
图6 51个免疫和肾结构细胞亚簇在每个样本的细胞比例条形图。
具体实施方式
以下将结合实施例对本申请的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本申请的目的、特征和效果。显然,所描述的实施例只是本申请的一部分实施例,而不是全部实施例,基于本申请的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本申请保护的范围。
试验方法中购入商品,品牌标注在试剂名称后的括号内,如中未注明具体条件者,按照常规条件或制造商建议的条件进行,所使用的试剂或仪器未注明生产厂商者,均可以通过市售购买获得的常规产品。
除非本文另有定义,否则结合本发明公开使用的科学和技术术语应具有本领域普通技术人员通常理解的含义,以下描述示例性方法和材料,但是与本文描述的那些类似或者等同的方法和材料也可以用于本发明的实践和测试中。
本文使用的以下词语和术语应具有所示出的含义:
在本文中所使用的术语“预后评估”、“诊断”或“监测”是指从医学的角度对人们的精神和体质状态做出判断,具体来说是一种确定哪种疾病或病症可以解释受试者的症状和体征的过程,例如,通过测定本文中所公开的标志物水平来确定受试者中肾脏疾病的存在,对肾脏疾病分期,判断肾病的严重程度,确定肾病的具体类型和阶段。
如本文所使用的,当可以使用句子中列出的事项中的“至少一种或多种”时使用“或”。当在本文中明确地描述为“在两个值”的“范围内”时,该范围还包括这两个值本身。
本文引用的参考文献诸如科学文献、专利和专利申请通过引用并入本文,其程度与具体描述每个文献相同。
本文所用,“阈值”是指针对特定变量值确定的值,其中当变化值大于或小于该值时,该值给出某种含义。阈值在此也称为界限值。
在本文中,术语“定量检测标志物”与鉴定/测量在受试者的样品中的存在/数量/水平/比例为等同意思。
在本文中,术语“iPT”特指受损的近端肾小管上皮细胞;“PT”指近端肾小管上皮细胞。
cDC2(2型常规树突状细胞),所述cDC2的标记基因为CD1c。
进一步的,所述标志物还包括iPT(SOX9和/或VCAM1标记)和/或Th1(IFNG标记)、Th17(IL17A标记)细胞。
本发明提供的标志物包括cDC2(CD1c标记)。
基于本发明的实验结果,作为一个具体的实施例,可以是cDC2(CD1c标记)单独作为标记物,当然可以理解的是cDC2(CD1c标记)也可以和现有技术中的其他标记物如尿蛋白肌酐比(尿蛋白/肌酐比值的全称是尿蛋白/肌酐比值测定,是用于监测尿蛋白排出情况的一种新的可靠方法)、狼疮肾炎慢性指数评分和尿TGF-β等指标协同使用。
作为另一个具体的实施例,可选的,可检测以下标志物组合用于狼疮性肾炎的综合评价:
cDC2(CD1c标记)、iPT(SOX9标记)组合;
cDC2(CD1c标记)、iPT(VCAM1标记)组合;
cDC2(CD1c标记)、iPT(SOX9和VCAM1标记)组合;
cDC2(CD1c标记)、Th1(IFNG标记)、Th17(IL17A标记)组合;
cDC2(CD1c标记)、iPT(SOX9标记)、Th17(IL17A标记)组合;
cDC2(CD1c标记)、iPT(SOX9标记)、Th1(IFNG标记)、Th17(IL17A标记)组合。
如上所述,显示了优选的实施方案以便于理解。本发明的范围不限于本文具体描述的实施方案和实施例,并且仅受权利要求的范围限制。下文根据试验过程具体的展示本发明的实施例。
实施例1:LN肾脏中单细胞图谱和疾病相关联细胞群的鉴定
为了解构LN肾脏中的微环境并鉴定与疾病相关联的特定细胞类型,我们通过对来自40例LN患者和6例器官供体健康肾脏的肾活检样本进行了单细胞RNA测序(scRNA-seq),获得了LN肾脏的高分辨率转录组学细胞图谱(图1a)。此外,从7例LN患者中收集外周血样本并进行单细胞RNA测序。经过严格的质量控制后,从肾活检样本中获得了总共268, 942个细胞,其中包括75, 643个CD45+免疫细胞和193, 299个CD45-肾脏固有细胞,用于后续分析。用统一流形近似和投影(UMAP)进行的初始细胞聚类和降维基于其典型标记表达鉴定出6个主要细胞区室[1,2],包括B细胞、T细胞、髓样细胞、内皮细胞、上皮细胞和间充质细胞(图1b,图4)。
通过对所有细胞进行无监督聚类得到51簇细胞。基于细胞特征基因表达对每簇细胞进行注释(图1b,图5)。每个簇中的细胞均来自多个样本(图6)。下文中列举了部分细胞,细胞名称结构为:细胞大群编号-细胞类型名字-代表基因,如cB01_naive_TCL1A,cB01为B细胞编号,naive是指幼稚B细胞,TCL1A为幼稚B细胞的代表基因。具体的,B细胞含有6个簇,包括cB01_naiveB_TCL1A、cB02_memoryB_CD27、cB03_activatedB_IER2、cB04_ABC_FCRL5、cB05_Bcell_MT1G和cB06_plasma_MZB1。T细胞由14个簇组成,其中5簇为CD4+T细胞(cT01_Tcm_CD4、cT02_Th1_IFNG、cT03_Th17_IL17A、cT04_Tfh_CXCR5和cT05_Treg_FOXP3),4个簇是CD8+T细胞(cT06_Tcm_CD8、cT07_Tem_GZMK、cT08_CTL_GZMB和cT09_MAIT_SLC4A10)、自然杀伤T细胞(cT12_NK_NKT)、γ-δT(cT13_gdT)、先天性淋巴细胞(cT14_ILC)以及具有高表达MT1G(cT10_Tcell_MT1G)和MKI67(cT11_Tcell_MKI67)的T细胞。髓样细胞由12个簇组成,包括5个巨噬细胞亚簇(cM08_Macro_IL1B、cM09_Macro_C1QA、cM10_Macro_MT1G、cM11_Macro_MKI67和cM12_Macro_SPP1)、3个DC亚簇(cM01_cDC1_CLEC9A、cM02_cDC2_CD1C和cM03_pDC_LILRA4)和2个单核细胞亚簇(cM06_Mono_CD14和cM07_Mono_CD14CD16)、肥大细胞(cM04_Mast_KIT)和中性粒细胞(cM05_Neut_FCGR3B)。在肾结构区室内,我们鉴定了肾小球内皮细胞(cEndo01_GE_EDH3)、直小血管内皮细胞(cEndo02_VR_PLVAP)、小静脉内皮细胞(cEndo03_VE_SOX17)和淋巴管内皮细胞(cEndo04_LE_MMRN1)、近端肾小管上皮细胞(PT)(cEpi03_PT_ALDOB)、Henle环上皮细胞(cEpi05_LOH_UMOD)和远曲肾小管上皮细胞(cEpi06_DCT_SLC12A3)、足细胞(cEpi01_Podo_NPHS1)、集合管两簇闰细胞(cEpi08_ICA_SLC4A1和cEpi09_ICB_SCL26A4)、集合管主细胞(cEpi07_PC_AQP2)、成纤维细胞(cMes01_Fib1_DCN和cMes02_Fib2_TNC)、周细胞(cMes04_Pe_RGS5)、血管平滑肌细胞(cMes05_vSMC_ACTA2)和系膜细胞(cMes03_Mesa_GATA3),还检测到高表达损伤和炎症相关基因的足细胞和近端小管上皮细胞,并因此分别被鉴定为受损的足细胞(cEpi02_ipodo_CDH6)和受损的PT(iPT)细胞(cEpi04_iPT_SOX9)。
与健康肾脏相比,LN肾脏中B细胞、T细胞、髓样细胞和间充质细胞的比例显著升高,而上皮细胞的比例较低(图1c-d)。此外,T细胞和髓样细胞的比例与疾病严重程度相关,在病理类型为IV/IV+V级LN患者中得分最高(图1e)。我们进一步分析了每种细胞类型与临床参数之间的相关性:Th17(cT03_CD4_IL17A)细胞、2型常规树突状细胞(cDC2)(cM02_cDC2_CD1C)和iPT细胞(cEpi04_iPT_SOX9)与24小时尿蛋白(24h-Upro)呈正相关,与估计肾小球滤过率(eGFR)呈负相关(图1f),表明它们与LN疾病严重程度相关,即Th17、cDC2和iPT细胞比例越高,肾脏病变越严重。值得注意的是,在这些细胞类型中,cDC2与24h-Upro和eGFR的相关性最强。与此同时,我们还观察到cT02_Th1_IFNG、cM04_Mast_KIT细胞比例与24h-Upro呈正相关,cEpi02_ipodo_CDH6与eGFR呈负相关,cM09_Macro_C1QA与24h-Upro呈负相关且与eGFR呈正相关(图1f)。
实施例2:LN肾脏中致病性DC3的鉴定
cDC2是前哨细胞,在启动和维持适应性免疫反应中发挥关键作用。它们可被细分为表型和功能异质性亚群。因此,我们对cDC2进行了亚聚类。cDC2再进行无监督聚类后产生了四个亚簇(图2a)。经典cDC2标记物,包括CD1C、FCER1A、CLEC10A,在亚簇C0和C2中表达,而CD163也在C0中显著表达(图2b)。C2和C0的转录谱分别类似于新定义的DC2和DC3亚群,因此被注释为DC2和DC3。相比之下,单核细胞基因C5AR1(CD88)的表达仅限于亚簇C1,以及CLEC10A和CD1C的表达,表明它们是单核细胞衍生的DC(mo-DC)。在这些cDC2亚簇中,只有DC3的比例同时与24h-Upro正相关(R=0.65,P=1.3x10-5),以及与eGFR负相关(R=-0.37,P=0.018)(图2c-d),这说明DC3可能是cDC2参与LN发病的关键成分。通过多重免疫组化(mIHC)染色和独立活检样本的流式细胞术表型分析,进一步证实了DC3在LN肾脏中的存在(图2e-f)。我们流式细胞学的门控策略如下:首先圈出活的单个细胞,然后圈出免疫细胞(CD45+),从免疫细胞中圈出髓系细胞(CD3-CD19-CD56-),排除单核细胞(CD88-),圈出经典树突状细胞(cDCs)(CD11C+HLA-DR+),再从cDCs中圈出CD1C+的cDC2,最后从cDC2中圈出CD163+的DC3(图2e)。
实施例3:肾脏DC3预测LN患者的治疗效果
肾脏DC3在疾病严重程度中的临床意义促使我们研究DC3浸润程度是否与LN患者的治疗效果相关联。在本研究中,在肾活检后接受免疫抑制剂联合糖皮质激素诱导疗法的LN患者中,13例患者完全缓解,6例患者未完全缓解。不完全缓解患者中的肾脏DC3比例显著较高(图3a)。我们还比较了不同缓解组之间Th1和Th17细胞的比例,因为它们是与疾病严重程度相关的两个其他细胞群,并观察到相同的趋势。然而,当我们通过肾活检石蜡切片的mIHC染色在独立LN队列中验证这些发现时,在不完全缓解的患者中,只有DC3显著富集(图3b-c)。为了进一步检验肾脏DC3在治疗效果中的预测能力,首先进行了使用人口统计学特性、临床病理学参数、肾脏中的DC3、Th1和Th17细胞计数的单变量分析。24h-Upro、外周血白细胞计数、血小板计数、肾脏病理中肾小管坏死、mIHC染色中Th1细胞计数和DC3计数与治疗无效性呈正相关,而eGFR与治疗无效性呈负相关(图3d)。此外,对这些变量的受试者操作特性(ROC)曲线进行比较,发现DC3计数具有最高的曲线下面积(AUC)0.84(图3e)。在多因素logistic回归分析中,仅肾脏中的DC3计数具有统计学差异(图3f)。这些结果强调肾脏DC3是接受诱导疗法的LN患者治疗效果的预测标志,其可用于临床实践中的患者分层。
本文中“[ ]”内的数字表征引用的参考文献,具体本文中涉及的参考文献如下:
1、Arazi, A., et al.,The immune cell landscape in kidneys of patients with lupus nephritis.Nat Immunol, 2019.20(7): p. 902-914.
2、Stewart, B.J., et al.,Spatiotemporal immune zonation of the human kidney.Science, 2019.365(6460): p. 1461-1466.
试验方法
样本采集
肾活检样本收集自在五个临床中心接受诊断性肾活检的LN患者。有两个独立的队列;一个是儿童LN的随机对照试验(ChiCTR2100053545),而另一个是成人LN的前瞻性队列。正常人肾组织从供肾移植前肾穿刺活检获得。该研究得到了中山大学附属第一医院机构审查委员会的批准,并获得了所有患者的知情同意。
将所有肾活检样本在采集后置于MACS®组织储存溶液(Miltenyi Biotec)中,并在2至3小时内新鲜处理用于测序。
组织加工和单细胞解离(参考:文献Arazi, A., et al.,The immune cell landscape in kidneys of patients with lupus nephritis.Nat Immunol, 2019. 20(7): p. 902-914. +联川生物公司提供的方法;)
新鲜肾活检标本切片约1 mm3,并在消化前用磷酸盐缓冲盐水(PBS,Gibco)洗涤2至3次。将切片和洗涤后的样本放入5 mL离心管中,用多组织解离试剂盒(MiltenyiBiotec)的2.5 mL消化酶溶液消化,并在振动筛(125 r.p.m)上于37℃下孵育30分钟,用3mL移液管每10分钟上下吸取悬浮液5至10次,以促进细胞解离。消化后,所得单细胞悬浮液通过30 μmMACS®智能过滤器(Miltenyi Biotec)过滤,残余组织用PBS(Gibco)洗涤2至3次,并且悬浮液也过滤,将两种悬浮液收集在15 mL锥形管中,于4℃下以400 g离心6分钟。沉淀物用200 μL PBS(Gibco)再悬浮,并与2 mL红细胞(RBC)裂解缓冲液(eBioscience™10X RBC裂解缓冲液)于4℃下孵育5分钟。RBC裂解后,悬浮液以400 g于4℃下离心6分钟,沉淀物用RPMI-1640培养基(Invitrogen)再悬浮以进行进一步操作。使用双荧光AO/PI方法,通过自动细胞计数器(CountstarRigel)对产生的单细胞进行定量和生存力分析。通过这种方法产生的单细胞悬液的存活力大于80%。
外周血单核细胞的分离(参考:密度梯度离心法分离人外周血单个核细胞)
血样首先用PBS(Gibco)稀释至1:2,然后在50 mL锥形管中用15 mL Ficoll-Paque小心分层,并于室温下以1800 r.p.m离心30分钟并制动。离心后,抽吸外周血单核细胞(PBMC)层并用PBC(Gibco)洗涤两次。
多重免疫组织化学染色(染色参考:IHC一抗染色方法+PANO试剂盒提供的染色方法;病理切片扫描和分析参考:TissueGnostics公司提供的扫描仪及分析软件)
根据制造商的方案,使用PANO 7-plex IHC试剂盒(Panovue)对4至5 μm福尔马林固定的石蜡包埋(FFPE)肾活检切片进行多重免疫组织化学(mIHC)染色。载玻片在二甲苯中脱蜡,并用100%、95%、75%乙醇和双蒸馏水再水合。通过柠檬酸盐缓冲液(pH 6.0)回收抗原,并在微波中加热至沸腾约20分钟,然后于室温下用5%牛血清白蛋白(BSA)封闭切片10分钟。依次应用抗CD163(abcam,ab182422)、抗CD11c(abcam,ab52632)和抗CD4(abcam,ab133616)、抗SLC22A6(abcam,ab135924)、抗VCAM1(abcam,ab134047)抗体。一级抗体于37℃下孵育30分钟,与辣根过氧化物酶结合的二级抗体于室温下孵育10分钟。用1:200的5%BSA双荧光团Opal 520、540、570、620和650进行酪酰胺信号扩增,并于室温下孵育10分钟。一级抗体染色后,用DAPI对细胞核进行染色。使用TissueFAXS平台(TissueGnostics)扫描染色载玻片,并使用StrataQuest软件(Tissue gnostics)处理图像。
采用抗体:
抗体 | 品牌 | 货号 | 克隆号 |
抗CD11c | Abcam | ab52632 | EP1347Y |
抗CD163 | Abcam | ab182422 | EPR19518 |
抗CD4 | Abcam | ab133616 | EPR6855 |
抗SLC22A6 | Abcam | ab135924 | / |
抗VCAM1 | Abcam | ab134047 | EPR5047 |
mIHC染色切片上的细胞定量(参考:TissueGnostics公司提供的扫描仪及分析软件)
根据“多重免疫组织化学染色”中描述的程序对肾活检切片进行mIHC染色。应用的抗体为抗CD163(abcam,ab182422)、抗CD11c(abcam、ab52632)、抗CD4(abcam和ab133616)、抗IFNG(abcam-ab231036)和抗IL17((R&D系统,AF-317-NA)。用StrataQuest软件(TissueGnostics)进行细胞定量分析。计算整个载玻片中DC3(CD11c+CD163+)、Th1(CD4+IFNG+)和Th17细胞(CD4+IL17+)的总数。
文库制备和scRNA-seq(由联川公司进行测序和文库制备)
使用Chromium Next GEM单细胞5’试剂盒v2(10X基因组学)按照制造商的方案进行乳液中凝胶珠的生成和条形码、cDNA扩增、5’基因表达文库构建、cDNA的V(D)J扩增和V(D)J文库构建。用生物分析仪高灵敏度芯片(Agilent)对构建的V(D)J富集和5’基因表达文库进行定量和评估。两个库均包含标准Illumina配对末端构建体,以P5开始,以P7结束,并且包括在读段1开始时编码的16 bp 10x条形码。样本索引序列作为i7索引读段并入。最终文库在NovaSeq 6000(Illumina)上测序,具有150 bp配对末端读段。
scRNA-seq数据的质量控制(使用10x Genomics提供的Cell Ranger单细胞软件分析)
使用由10x Genomics提供的Cell Ranger单细胞软件套件(v5.0.1)对原始scRNA-seq数据进行预处理,用于解复用细胞条形码、读段比对和在GRCh38人类参考基因组下生成基因-细胞矩阵。Seurat R软件包(v4.0.5)生成并评估了详细的QC指标。在少于3个细胞中检测到的基因和其中检测到的转录物少于200或多于8000个基因,或大于70%的UMI来源于线粒体基因或log10基因计数/log10UMI计数>0.80的细胞被滤出,并从后续分析中排除。由于免疫细胞和肾驻留细胞之间线粒体含量的差异,UMI < 15%来源于线粒体基因的免疫细胞、UMI < 30%来源于粒线基因的肾驻留细胞(近端肾小管细胞除外)被纳入进一步分析。对于主要细胞类型的亚聚类,检测到的基因小于500的细胞被进一步去除,但髓样细胞除外,其中检测到的基因<200的截止值被保留,以避免去除中性粒细胞。通过簇标记基因表达鉴定双倍体:一个簇的细胞表达来自两个或多个不同细胞谱系的标记(例如PTPRC和EPCAM、CD3D和CD79A)。我们仔细审查了典型标记基因的表达,并重复了上述步骤几次,以确保我们已经去除了与细胞双倍体相关联的大多数条形码。然后我们去除了细胞质基因,诸如线粒体、核糖体和血红蛋白基因。
细胞聚类和注释(使用Seurat R包进行数据分析,并参考文献进行注释)
在去除劣质细胞和双倍体后,Seurat R包(v4.0.5)应用于基因计数矩阵归一化、缩放和具有默认参数的高度可变基因鉴定。主成分(PC)由ElbowPlot函数鉴定。前2000个可变基因和前25个PC用于非监督聚类分析,分辨率设定为0.1。我们基于典型细胞类型特异性标记物鉴定出六种主要细胞类型,包括T细胞(CD3E)、髓样细胞(LYZ)、B细胞(CD79A)、肾上皮细胞(EPCAM)、内皮细胞(PECAM1)和间充质细胞(PDGFRB)。使用适当调整的参数对每个主要细胞类型进行第二轮亚聚类,以鉴定出主要细胞类型内的亚簇和细胞亚型注释。为了可视化,使用具有Seurat的RunUMAP函数的UMAP方法降低维数。经由FindAllMarkers函数鉴定簇特异性标记基因,这些标准如下:1)only.pos = TRUE,2)min.pct = 0.25,3)log FC>0.25。
统计分析
除上述用于scRNA-seq数据分析的生物信息学方法外,所有其他统计分析均使用统计软件R v4.0进行。使用非配对双尾Wilcoxon秩和检验分析两组的细胞比例。进行Pearson相关分析以评估两个连续变量之间的关系(例如,细胞比例与临床病理学表型)。结果在p值<0.05时具有统计学意义。
Claims (10)
1.定量检测标志物的试剂在制备用于预后评估、诊断或监测狼疮性肾炎试剂盒中的应用,其特征在于,所述标志物为cDC2,所述cDC2的标记基因为CD1c。
2.根据权利要求1所述的应用,其特征在于,所述标志物还包括iPT(受损的近端肾小管上皮细胞)和/或Th1、Th17细胞。
3.根据权利要求2所述的应用,其特征在于,所述Th1细胞的标记基因为IFNG。
4.根据权利要求2所述的应用,其特征在于,所述Th17细胞的标记基因为IL17A。
5.根据权利要求2所述的应用,其特征在于,所述iPT(受损的近端肾小管上皮细胞)细胞的标记基因为SOX9和/或VCAM1。
6.根据权利要求1-5任一所述的应用,其特征在于,用于预后评估、诊断或监测狼疮性肾炎的方法为,测定在受试者的样品中所述标志物至少一种的数量,其中所述数量与所述受试者中狼疮性肾炎的严重程度呈正相关。
7.根据权利要求6所述的应用,其特征在于,所述方法为当在测试样品中标志物的数量大于在来自相同受试者的早期样品中的数量时,鉴定出在受试者疾病的恶化,当在测试样品中标志物数量低于在早期样品中的数量时,鉴定出在受试者中疾病的改善。
8.治疗狼疮性肾炎的药物筛选方法,其特征在于,所述方法为在狼疮性肾炎患者服用药物前后,测试同一患者样品中权利要求1-5任一所述标志物的数量,当样品中标志物的数量低于服药前的数量,鉴定出在受试者中疾病的改善,药物有效。
9.根据权利要求8所述的治疗狼疮性肾炎的药物筛选方法,其特征在于,所述药物为免疫抑制剂和/或糖皮质激素。
10.根据权利要求7所述的应用或权利要求8或9所述的方法,其特征在于,所述样品为肾活检穿刺获得的肾皮质和/或髓质。
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Application publication date: 20230829 |