CN116400075B - 检测狼疮性肾炎标志物的试剂及方法 - Google Patents
检测狼疮性肾炎标志物的试剂及方法 Download PDFInfo
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Abstract
本发明属于多重免疫组化技术领域,具体涉及一种检测狼疮性肾炎标志物的试剂及方法,所述标志物为DC3细胞,所述试剂包括抗CD163抗体与抗CD11c抗体、抗CD1c抗体中的至少一种,本发明操作方便、简单,能在较短的时间内检测LN患者肾脏DC3细胞的数量,对其浸润程度进行评估;特异性好,本发明根据DC3细胞的特征基因设计抗体,可准确对LN患者肾脏DC3细胞进行定量检测,具有较高的特异性。
Description
技术领域
本发明属于多重免疫组化技术领域,具体涉及一种检测狼疮性肾炎标志物的试剂及方法。
背景技术
狼疮性肾炎(lupus nephritis, LN)是系统性红斑狼疮(systemic lupuserythematosus,SLE)最常见且严重的并发症。治疗未达缓解的LN患者中有10~30%会进展至终末期肾病,严重时危及生命,故准确预测LN患者疗效在指导临床、制定个体化治疗方案中至关重要。然而,目前尚无公认的预测LN患者诱导缓解治疗疗效的临床、病理和生物学指标。近年来,有研究采用肾间质炎症、纤维化和透明血栓等肾脏病理特征联合尿蛋白肌酐比、估算肾小球滤过率、血清抗双链DNA抗体和补体3水平,以及血管细胞粘附分子1、趋化因子配体2等指标构建LN诱导缓解治疗疗效的预测模型,但因样本量小、异质性大、缺乏外部验证或需多个预测指标联合等因素限制,尚未能达到临床应用水平。
通过对LN患者肾穿刺样本进行scRNAseq,深度剖析了LN肾脏的微环境,我们发现:LN肾脏中DC3是与疾病严重程度相关的细胞亚群;DC3可以作为预测LN患者诱导治疗疗效的标记物;DC3具有很强的促炎能力,可通过分泌细胞因子、提呈抗原的作用募集、激活多种CD4+T细胞,驱动免疫反应并造成组织损伤;肾脏损伤的小管上皮细胞可以分泌趋化因子和粘附分子促进DC3的募集和粘附,促使更多DC3停留在小管间质激活T细胞,聚集形成炎症浸润灶,加重肾脏损伤,为LN的发病机制提供了新见解,为治疗策略的制定及疗效的预测提供了新的方向。
尽管免疫组织化学染色及成像分析是研究组织形态和组织原位抗原表达不可或缺的检测技术,广泛应用于临床病理诊断等领域,但是受制于传统单标记免疫组织化学染色方法的限制,通常只能对组织中的一到两种抗原进行染色分析传统的免疫组织化学染色或免疫荧光染色。而新的mIHC技术能够在同一张组织切片样本上同时检测多种靶标分子,获得关于特殊细胞定量和空间排列的多通道信息,而且配备专业分析软件,具有高重复性、高效率和高成本效益的优势,而根据mIHC检测的原理,抗体的选择极大的影响了检测的灵敏性和特异性,现未有针对肾脏中DC3细胞多重免疫组化检测方法,并用于将mIHC应用于预测LN治疗效果。
发明内容
本发明的主要目的是针对背景技术存在的问题,提供一种检测狼疮性肾炎标志物的试剂及方法,为狼疮性肾炎诊标志物的检测提供一种解决方案。
为实现以上目的,本发明提供以下技术方案:
检测狼疮性肾炎标志物的试剂,所述标志物为DC3细胞,所述试剂包括抗CD163抗体与抗CD11c抗体、抗CD1c抗体中的至少一种。
优选的,所述试剂包括抗CD11c抗体和抗CD163抗体。
本发明还提供了一种试剂盒,包括上述抗体的试剂。
可以理解的是,在本发明提供检测试剂下,可采用多种检测方法对本发明中的标志物DC3细胞进行检测,可供选择的如免疫荧光技术(IF)、免疫组织化学技术(IHC)、流式细胞术等。
作为一个具体的实施例,本发明提供了一种多重免疫组化检测用试剂盒,所述试剂盒包括:
一抗:Anti-CD11c抗体、Anti-CD163抗体;HRP二抗:HRP标记二抗;TSA荧光染料;DAPI染色液。
可以理解的,TSA荧光染料可以有多种选择,包括单色荧光染料-520、单色荧光染料-650、单色荧光染料-480、单色荧光染料-520、单色荧光染料-570、单色荧光染料-620、单色荧光染料-650、单色荧光染料-780,优选的,本发明使用的 TSA荧光染料为单色荧光染料-520、单色荧光染料-650。
进一步的,本发明还提供了一种多重免疫组化法检测肾脏中DC3细胞的方法,包括以下步骤:
(1)样品准备:肾穿刺活检石蜡组织切片脱蜡、复水;(2)抗原修复;(3)封闭抗体;(4)染色:用1X PBS清洗切片,加入Anti-CD11c抗体,37℃孵育,清洗后加入HRP二抗,室温孵育,再次清洗后加入TSA荧光染料,室温孵育;(5)Anti-CD163抗体染色:重复步骤(2)、(3)、(4);(6)DAPI染色、封片、扫片;(7)通过分析系统检测并统计完整视野中DC3(CD11c+CD163+)细胞的数量。
优选的,所述步骤(2)具体为:将切片浸入修复液,再放入微波炉中加热至沸腾,沸腾后继续保持95℃约10-15分钟,冷却;步骤(4)中Anti-CD11c抗体的稀释比例为1:200;步骤(5)中Anti-CD163抗体的稀释比例为1:500。
优选的,所述步骤(2)具体为:将切片浸入修复液,再放入微波炉中加热直至沸腾。
可以理解的是,抗原修复液可以有多种选择,如OEDTA或柠檬酸盐修复液等,优选的,所述修复液为柠檬酸盐修复液。
进一步的,所述抗体的制备步骤为:
S1:杂交瘤细胞的制备:采用目的抗原免疫小鼠,使小鼠产生致敏B淋巴细胞;
S2:细胞融合:收集小鼠骨髓瘤细胞和脾细胞并进行前处理,处理后按比例添加,进行细胞融合;
S3:杂交瘤细胞的培养和筛选:将融合细胞转移至筛选培养基中,在培养箱中培养数天后进行杂交瘤抗体筛选;
S4:单克隆抗体的大量制备:将制备好的杂交瘤细胞接种在小鼠腹腔中,等待数周,见小鼠腹部膨大,用注射器抽取腹水,即可获得大量单克隆抗体。
进一步的,所述抗CD163抗体制备时使用的目的抗原氨基酸序列如SEQ ID No.1所示,和/或所述抗CD11c抗体制备时使用的目的抗原氨基酸序列如SEQID No.2所示,和/或所述抗CD1c抗体制备时使用的目的抗原氨基酸序列如SEQ ID No.3所示。
进一步的,上述试剂或试剂盒及方法可用于狼疮性肾炎诊断中的使用,用于检测标志物DC3的数量从而判断疾病的严重程度。
本发明操作方便、简单,能在较短的时间内检测LN患者肾脏DC3细胞的数量,对其浸润程度进行评估;特异性好,本发明根据DC3细胞的特征基因设计抗体,可准确对LN患者肾脏DC3细胞进行定量检测,具有较高的特异性。本发现实现了在诊断LN时准确预测患者对诱导治疗的缓解情况,精准定位采用标准一线诱导治疗方案无法达到完全缓解的LN患者,为临床诊疗提出决策支持,适合推广应用。
附图说明
图1、来源于健康供肾和LN患者的肾活检样本的cDC2的UMAP显示图(共有4个亚簇)。
图2、显示标记基因表达的cDC2的UMAP显示图。
图3、显示LN患者中每个cDC2亚簇的比例(相对于髓样细胞总数)与24UPO和eGFR之间的Pearson相关性的点图。
图4、显示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。
图5 、LN肾脏中DC3的流式细胞术圈门策略图;(DC3被定义为活的、单一的、LIN(CD56、CD3、CD19-)CD88-HLA-DR+CD11C+CD1C+CD163+细胞)。
图6、抗CD11c和CD163的LN患者肾活检切片的代表性mIHC染色示例图;(白色箭头表示DC3;原始放大倍数,20倍;比例尺,50 μm)。
图7、 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。)。
图8、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。)。
图9、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显示图(原始放大倍数,20倍;比例尺,50 μm。)。
图10、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。)。
图11、DC3数目、24hUpro、WBC、eGFR和肾小管坏死的单变量逻辑回归模型的ROC曲线图(WBC,白细胞计数。)。
图12、棒棒糖图(显示LN患者CR和NCR之间的多变量分析。)。
具体实施方式
下面结合附图和具体实施方式进一步说明本发明,这些附图和实施例仅作为说明本发明的例子,不限制本发明的范围。下述实施例中的实验方法,如无特殊说明,均为常规方法。试验方法中的购入商品,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。
基于本发明前期的研究,本发明设计了LN疾病中检测DC3的试剂,作为实施例,该试剂包括抗CD163抗体与抗CD11c抗体;或抗CD1c抗体与抗CD163抗体,这两种组合的试剂可用于多种检测方法,检测本发明中的标志物DC3,来用于LN的诊断、预后评估等应用。
实施例1
根据新型免疫细胞亚群DC3细胞的特征基因设计2种特异性一抗(与DC3细胞上特异性抗原结合的蛋白),分别是Anti-CD11c抗体、Anti-CD163抗体;利用mIHC技术对LN患者肾穿刺活检石蜡组织切片进行染色,通过全景组织多光谱成像仪进行扫描成像,再利用分析系统进行全景组织微环境中DC3细胞的精准检测和定量分析。
多重免疫组化法检测肾脏中DC3细胞的方法,具体包括如下步骤:
mIHC染色抗体及荧光染料的准备:
(1)一抗:Anti-CD11c抗体、Anti-CD163抗体
(2)二抗:HRP标记山羊抗兔IgGH&L(abcam,ab6721)
(3)TSA荧光染料:单色荧光染料-520、单色荧光染料-650
(4)DAPI染色液
用上述抗体和荧光染料按以下方法对LN患者肾穿刺活检石蜡组织切片进行染色。
本实例的组织切片为已诊断为LN的患者肾穿刺活检石蜡组织切片。
1.肾穿刺活检石蜡组织切片脱蜡、复水;
2.抗原修复:将切片浸入1X柠檬酸盐修复液,再放入微波炉中高火加热3-5分钟直至沸腾,沸腾后继续低火保持95℃约10-15分钟,室温冷却30分钟;
3.封闭抗体:用1X PBS清洗切片3次(每次5分钟)后,加入10%山羊血清(Solarbio,SL038),室温孵育10分钟;
4.染色:用1X PBS清洗切片3次(每次5分钟)后,加入Anti-CD11c抗体(1%山羊血清稀释,稀释比例:1:200),37℃孵育30min;清洗后加入HRP二抗(1%山羊血清稀释,稀释比例1:1000),室温孵育10min;再次清洗后加入单色荧光染料-520(信号放大反应液稀释,稀释比例:1:100),室温孵育10min;
5.重复步骤2至4,采用Anti-CD163抗体(1%山羊血清稀释,稀释比例:1:500)和单色荧光染料-650(TSA信号放大液稀释,稀释比例:1:100)染色;
6.DAPI染色:用1X PBS清洗切片3次(每次5分钟)后,加入DAPI工作液(双蒸水稀释,稀释比例1:100),室温孵育10min;
7.封片:用1X PBS清洗切片3次(每次5分钟)后,加入抗淬灭封片剂,加盖玻片,封片;
8.扫片:采用全景组织多光谱成像仪对完成染色的切片进行扫描,构建高维数字化信息图像;
9.DC3细胞定量检测:通过专业分析系统对数字化切片进行全视野分析,检测并统计完整视野中DC3(CD11+CD163+)细胞的数量。
本实例LN患者肾穿刺活检石蜡组织切片效果如图6所示,具有较高的特异性。
实施例2
与实施例1的其他步骤相同,除:一抗为Anti-CD1c抗体、Anti-CD163抗体。
实施例1和实施例2中的试剂可以由市场购买,也可以自行制备,具体的由市场购买,抗体可选的如重组Anti-CD11c抗体[EP1347Y]、重组Anti-CD163抗体[EPR19518]、Anti-CD1c抗体[EPR23189-305]。抗体也可以自行制备,具体见以下实施例。
实施例3
Anti-CD163抗体制备方法:
一、动物免疫
采用目的抗原: CD163胞外域氨基酸蛋白片段,氨基酸序列如SEQ ID No.1所示,免疫小鼠,使小鼠产生致敏B淋巴细胞,具体步骤为:
1)选用6-8周龄雌性BALB/c小鼠,用0.5ml注射器吸入CD163抗原与弗氏完全佐剂按比例1:1混合乳化的免疫抗原,分3点,每点0.1ml左右,给小鼠进行背部(或腹部)皮下注射,共接种2-4次,每次间隔2-4周;
2)接种2-4次后采血进行抗体滴度ELISA检测,通过ELISA检测确定小鼠血中抗体阳性,小鼠即可用于后续的细胞融合和杂交瘤细胞制备:
ELISA检测方法(一抗为小鼠血液纯化抗体,二抗为HRP标记山羊抗鼠二抗),ELISA包括以下步骤:
a. 包被:用碳酸盐包被缓冲液将抗体稀释至浓度1-10μg/ml。在聚苯乙烯酶标板的每孔加200μl,4℃过夜。次日,弃去孔内溶液,用洗涤缓冲液洗3次,每次3min;
b.洗涤:移去包被液,用含0.05%吐温20的PBS溶液冲洗3次,每次3分钟;
c. 加样孵育:每孔加入1:200稀释的样本150ul,封板后置37℃孵育1小时;
e. 洗涤:用含0.05%吐温20的PBS溶液冲洗3次,每次3分钟;
f. 加抗体孵育:每孔加入200ul已稀释的酶标记抗体溶液,37℃孵育1.5h;
g. 洗涤:用含0.05%吐温20的PBS溶液冲洗3次,每次3分钟;
h. 加酶结合物孵育:每孔加入稀释好的酶结合物工作液100 μl,37℃避光孵育30min;
i. 洗涤:用含0.05%吐温20的PBS溶液冲洗3次,每次3分钟;
j. 加显色底物:每孔加入TMB-过氧化氢尿素溶液100μl,37℃避光反应10min;
k. 终止反应:每孔加入2M硫酸80μl并于20分钟内测定结果;
l. 结果判断:TMB反应后采用450nm波长检测,以空白对照孔调零后测各孔OD值。
二、细胞融合
收集小鼠骨髓瘤细胞和脾细胞并进行前处理,按照1:5比例将处理后的小鼠骨髓瘤细胞和脾细胞进行细胞融合,细胞融合方法为聚乙二醇(PEG)细胞融合,包括以下步骤:
1)将骨髓瘤细胞与脾细胞按1:5的比例混合在一起;
2)在50ml离心管中用无血清不完全培养液洗1次,1200r/min离心8分钟,弃去上清,用吸管吸净残留液体;
3)用吸管在1分钟内加预热至37℃的50%PEG(PH8.0)1ml,边加边轻轻搅拌;
4)用10ml吸管在90s内加20-30ml预热的基础培养基,用于终止PEG作用,室温静置10分钟;
5)1000r/min离心6分钟,弃去上清;
6)加入5ml血清重悬细胞,再加入5ml HAT培养基(Biological Industries,03-080-1B),轻轻吹打,使其悬浮并混匀,
7)分装96孔细胞培养板,每孔0.1-0.15ml,37℃,6% CO2培养箱内培养;
8)定期观察杂交瘤细胞的生长情况,待其长至孔底面积1/10以上时吸出上清供抗体检测。
三、杂交瘤细胞的培养和筛选
将融合细胞转移至HT筛选培养基(Biological Industries,03-085-1B)中,在二氧化碳培养箱中培养7~10天后进行杂交瘤抗体筛选,培养箱温度37℃,CO2浓度为6%;
1)用含有HT的完全培养基进行系列倍比稀释直至每毫升含有5,10,50个细胞,每孔接种0.1ml细胞悬液,即每孔分别含有0.5,1和5个细胞;
2)37℃,6%CO2培养箱内培养7-10天,出现肉眼可见的克隆或者倒置与显微镜下能够观察到杂交瘤细胞布满孔底1/5面积时就可以检测抗体,随后选择效价高,只有单个克隆生长的形态良好的细胞孔,继续进行1-2次克隆和扩大培养;将所得到的克隆细胞进行冻存。
四、单克隆抗体的大量制备
将制备好的杂交瘤细胞接种在BALB/c小鼠腹腔中,约1-2周,可见小鼠腹部膨大,用注射器抽取腹水,即可获得大量单克隆抗体,单克隆抗体的大量制备方法为体内诱生法,采用腹腔注射0.5ml液体石蜡或降植烷对BALB/c小鼠进行预处理,1-2周后,再向腹腔内接种杂交瘤细胞,取出的腹水于4℃离心4000rpm,10min。小心吸出中间的腹水收集于离心管中,4℃或-20℃保存。
五、抗体纯化
用HiTrap rProtein A FF(GE公司)亲和层析法按说明书从腹水中纯化抗体。SDS-PAGE胶鉴定纯度,Bradford法测定浓度。纯化的抗体保存于-20℃备用。
实施例4
其他步骤同实施例3一致,区别在于,采用目的抗原: CD11c胞外域氨基酸蛋白片段,氨基酸序列如SEQ ID No.2所示。
实施例5
其他步骤同实施例3一致,区别在于,采用目的抗原: CD1c胞外域氨基酸蛋白片段,氨基酸序列如SEQ ID No.3所示。
LN疾病中DC3的发现及作为标志物的效果验证试验
本发明基于先前的研究,由于cDC2在启动和维持适应性免疫反应中发挥关键作用。且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,以进一步揭示LN(狼疮性肾炎)疾病的罪魁祸首。cDC2再进行无监督聚类后产生了四个亚簇(图1)。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2)。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3、4、5),这说明DC3可能是cDC2参与LN发病的关键成分,检测DC3的数量指标对LN的严重程度分级有重大的意义,具体的,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5)。
同时本发明验证了肾脏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7)。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8-9)。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10)。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11)。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12)。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。
上述试验所用方法:
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 μm MACS®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™10XRBC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(Countstar Rigel)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%。
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(Tissuegnostics)处理图像。
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)。用StrataQuest软件(TissueGnostics)进行细胞定量分析。计算整个载玻片中DC3(CD11c+CD163+)的总数。
流式细胞术(参考:BD-Biosciences公司提供的染色方案)
根据“组织处理”中描述的程序制备单细胞悬浮液。对于表面染色,将在PBS(Gibco)染色缓冲液中的2%胎牛血清(FBS)中稀释的选定抗体添加到细胞中,并于室温下孵育20分钟。对于细胞因子的胞浆内染色,首先将细胞与含有BD GolgiPlug™(BDBiosciences)的白细胞活化混合物于37℃下孵育4-6小时。然后将细胞固定并用BDCytofix/Cytoperm ((BD-Biosciences)于4℃下渗透20分钟。将在BD Perm/WashTM(BD-Biosciences)中稀释的抗体添加到细胞中,并于4℃下孵育60分钟。染色后,洗涤细胞并用200 μL 1%多聚甲醛(PFA)溶液固定。使用SpectroFlo(CYTEK)对AURORA/NL进行流式细胞术,并使用FlowJo(TreeStar,版本10.4.0)分析数据。
采用抗体:
抗体 | 通道 | 品牌 | 货号 | 克隆号 |
FVS | AF700 | BD | 564997 | / |
CD45 | BUV395 | BD | 563792 | HI30 |
CD3 | BV510 | BD | 564713 | HIT3a |
CD19 | BV510 | BD | 562947 | SJ25C1 |
CD56 | BV711 | BD | 563169 | NCAM16.2 |
CD11c | APC-CY7 | BD Custom | 624355 | B-ly6 |
HLA-DR | BUV805 | BD | 748338 | G46-6 |
CD1c | PE-Cy7 | Biolegend | 331516 | L161 |
CD163 | BV421 | BD | 562643 | GHI/61 |
CD88 | BV786 | BD | 742320 | D53-1473 |
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。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
Claims (14)
1.检测狼疮性肾炎标志物的试剂在制备用于预测狼疮性肾炎接受免疫抑制剂联合糖皮质激素诱导疗法治疗效果产品中的应用,其特征在于,所述标志物为DC3细胞,所述试剂为Anti-CD163抗体与Anti-CD11c抗体或Anti-CD1c抗体中至少一种的组合。
2.根据权利要求1所述的应用,其特征在于,所述试剂为Anti-CD11c抗体和Anti-CD163抗体组合。
3.一种试剂盒在制备用于预测狼疮性肾炎接受免疫抑制剂联合糖皮质激素诱导疗法治疗效果产品中的应用,其特征在于,含权利要求1或2的试剂组合。
4.根据权利要求3所述的应用,其特征在于,所述试剂盒具体应用在多重免疫组化检测中,包括:一抗:Anti-CD11c抗体、Anti-CD163抗体;HRP二抗TSA荧光染料;DAPI染色液。
5.根据权利要求4所述的应用,其特征在于,所述TSA荧光染料包括单色荧光染料-520、单色荧光染料-650。
6.根据权利要求4所述的应用,其特征在于,包括以下步骤:
(1)样品准备:肾穿刺活检石蜡组织切片脱蜡、复水;
抗原修复;
封闭抗体;
染色:用1X PBS清洗切片,加入Anti-CD11c抗体,37℃孵育,清洗后加入HRP二抗,室温孵育,再次清洗后加入TSA荧光染料,室温孵育;
Anti-CD163抗体染色:重复步骤(2)、(3)、(4);
DAPI染色、封片、扫片;
通过分析系统检测并统计肾脏病理切片完整视野中DC3(CD11c+CD163+)细胞的数量。
7.根据权利要求6所述的应用,其特征在于,所述步骤(2)具体为:将切片浸入修复液,再放入微波炉中加热至沸腾,沸腾后继续保持95℃10-15分钟,冷却;步骤(4)中一抗采用1%山羊血清稀释,Anti-CD11c抗体稀释比例为1:200;步骤(5)中Anti-CD163抗体稀释比例为1:200。
8.根据权利要求7所述的应用,其特征在于,所述修复液为柠檬酸盐修复液。
9.根据权利要求1或2所述的应用,其特征在于,所述抗体的制备步骤为:
S1:杂交瘤细胞的制备:采用目的抗原免疫小鼠,使小鼠产生致敏B淋巴细胞;
S2:细胞融合:收集小鼠骨髓瘤细胞和脾细胞并进行前处理,处理后按比例添加,进行细胞融合;
S3:杂交瘤细胞的培养和筛选:将融合细胞转移至筛选培养基中,在培养箱中培养数天后进行杂交瘤抗体筛选;
S4:单克隆抗体的大量制备:将制备好的杂交瘤细胞接种在小鼠腹腔中,等待数周,见小鼠腹部膨大,用注射器抽取腹水,即可获得大量单克隆抗体。
10.根据权利要求9所述的应用,其特征在于,所述抗CD163抗体制备时使用的目的抗原氨基酸序列如SEQ ID No.1所示,和/或所述抗CD11c抗体制备时使用的目的抗原氨基酸序列如SEQ ID No.2所示,和/或所述抗CD1c抗体制备时使用的目的抗原氨基酸序列如SEQ IDNo.3所示。
11.根据权利要求9所述的应用,其特征在于,步骤S1中具体包括:
(1)选用6-8周龄雌性BALB/c小鼠,目的抗原与弗氏完全佐剂按比例混合,给小鼠进行皮下注射,共接种2-4次,每次间隔2-4周;
(2)接种2-4次后采血进行抗体滴度ELISA检测,通过ELISA检测确定小鼠血中抗体阳性后,小鼠即可用于后续的细胞融合和杂交瘤细胞制备。
12.根据权利要求9所述的应用,其特征在于,步骤S2中具体包括:按照1:5比例将前处理后的小鼠骨髓瘤细胞和脾细胞采用聚乙二醇介导进行细胞融合。
13.根据权利要求9所述的应用,其特征在于,步骤S3中具体包括:将融合细胞转移至HT筛选培养基中,在二氧化碳培养箱中培养7~10天后进行杂交瘤抗体筛选。
14.根据权利要求11所述的应用,其特征在于,所述抗体滴度ELISA检测方法:一抗为小鼠血液纯化抗体,二抗为HRP标记山羊抗鼠二抗,检测步骤依次为:包被、洗涤、加样孵育然后洗涤、加抗体孵育然后洗涤、加酶结合物孵育然后洗涤、加显色底物反应后,终止反应进行结果判断。
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