CN112080560B - Cd177在制备诊断胆道闭锁的产品中的应用 - Google Patents
Cd177在制备诊断胆道闭锁的产品中的应用 Download PDFInfo
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Abstract
本发明提供了一种CD177在制备诊断胆道闭锁的产品中的应用,涉及分子诊断技术领域,本发明通过检测大量的胆道闭锁患儿样本,并结合动物实验,首先证实了胆道闭锁外周血和肝脏组织中的CD177+中性粒细胞的数量可作为评估肝损伤程度的有力指标,因此检测CD177基因、CD177蛋白或CD177+中性粒细胞均可作为临床上评估胆道闭锁患儿的病情提供更方便、直接、准确的方法。本发明还证实了CD177在胆道闭锁中具有促炎的作用,其抑制剂可用于治疗胆道闭锁的药物研发。
Description
技术领域
本发明涉及分子诊断技术领域,尤其是涉及一种CD177在制备诊断胆道闭锁的产品中的应用。
背景技术
胆道闭锁(biliary atresia,BA)是以新生儿黄疸为主要表现的胆管阻塞性疾病,预后差,病死率高,病因及发病机制尚不明确。BA的基本病理特点为肝内外胆管进行性炎症、胆管闭锁和肝纤维化。其肝纤维化的进展比其他成人疾病发展快且侵袭性强,即使Kasai手术能缓解部分BA患儿的胆汁淤积症状,然而大多数患儿仍因后续的肝内胆管进行性破坏,甚至肝衰竭,成为危及患儿生命的严重疾病。我国是BA的高发地区,发病率高达1.5/10,000。由于目前缺乏相关的早期筛查方法及有效的临床治疗药物,BA患儿常常在出生后1-4月时才被确诊,大多数患儿因无法实施肝脏移植而于2岁内因肝衰竭而死亡。因此,寻找BA早期诊断方法极其重要。
目前胆道闭锁诊断方法较多,但特异性均较差,其方法包括:
(1)血清胆红素的动态观察:每周测定血清胆红素,如胆红素量曲线随病程趋向下降,则可能是肝炎;若持续上升,提示为胆道闭锁,但重型肝炎并伴有肝外胆道阻塞时,亦可表现为持续上升,此时则鉴别困难;
(2)超声检查:若未见胆囊或见有小胆囊(1.5cm以下),则疑为胆道闭锁,若见有正常胆囊存在,则支持肝炎,如能看出肝内胆管的分布形态,则更能帮助诊断;
(3)99mTc-diethyl iminodiacetic acid(DIDA)***试验:近年已取代131碘标记玫瑰红***试验,有较高的肝细胞提取率(48%~56%),优于其他物品,可诊断由于结构异常所致的胆道部分性梗阻,如胆总管囊肿或肝外胆管狭窄,发生完全梗阻时,则扫描不见肠道显影,可作为重症肝内胆汁郁积的鉴别,在胆道闭锁早期时,肝细胞功能良好,5分钟显现肝影,但以后未见胆道显影,甚至24小时后亦未见肠道显影,当新生儿肝炎时,虽然肝细胞功能较差,但肝外胆道通畅,因而肠道显影;
(4)脂蛋白-X(Lp-x)定量测定:脂蛋白-X是一种低密度脂蛋白,在胆道梗阻时升高,据研究所有胆道闭锁病例均显升高,且在日龄很小时已呈阳性,新生儿肝炎病例早期呈阴性,但随日龄增长也可转为阳性,若出生已超过4周而Lp-X阴性,可除外胆道闭锁;如>500mg/dl,则胆道闭锁可能性大,亦可服用消胆胺4g/天,共2~3周,比较用药前后的指标,如含量下降则支持新生儿肝炎综合征的诊断,若继续上升则有胆道闭锁可能;
(5)胆汁酸定量测定:最近应用于血纸片血清总胆汁酸定量法,胆道闭锁时血清总胆汁酸为107~294μmol/L,一般认为达100μmol/L都属郁胆,同年龄无黄疸对照组仅为5~33μmol/L,平均为18μmol/L,故有诊断价值,尿内胆汁酸亦为早期筛选手段,胆道闭锁时尿总胆汁酸平均为19.93±7.53μmol/L,而对照组为1.60±0.16μmol/L,较正常儿大10倍;
(6)肝穿刺病理组织学检查:一般主张作肝穿刺活检,或经皮肝穿刺造影及活检,新生儿肝炎的特征是小叶结构排列不整齐,肝细胞坏死,巨细胞性变和门脉炎症,胆道闭锁的主要表现为胆小管明显增生和胆汁栓塞,门脉区域周围纤维化,但有的标本亦可见到多核巨细胞,因此,肝活检有时能发生诊断困难,甚至错误,有10~15%病例不能凭此作出正确诊断;而且肝穿刺活检属于有创检查;
(7)胆道造影检查:胆道造影术是目前诊断胆道闭锁的“金标准”,造影诊断胆道闭锁有以下情况:①仅肝外胆管显影,肝内胆管不显影,提示肝内型闭锁;②肝内、外胆管均不显影,提示肝内外闭锁。新生儿肝炎综合征有下列征象:肝内外胆管均显影,胆总管显影,但较细。
由以上可知,虽然胆道造影检查是目前诊断胆道闭锁的“金标准”,但其创伤性太大,实施该检查的患儿年龄普遍偏大,且脱离手术治疗的最佳时机。其他检查方法的特异性均较差。临床上缺乏与胆道闭锁相关的诊断标志物,因此寻找胆道闭锁的诊断标志物更加有助于胆道闭锁患儿的诊断。
有鉴于此,特提出本发明。
发明内容
本发明的主要目的在于提供检测CD177的物质在制备用于诊断胆道闭锁的产品中的应用,以至少缓解了现有技术中存在的技术问题之一。
本发明提供了检测CD177的物质在制备用于诊断胆道闭锁的产品中的应用。
本发明还提供了检测CD177的物质在制备用于评估胆道闭锁患儿肝脏损伤程度的产品中的应用。
本发明还提供了检测CD177的物质在制备用于预后评估评估胆道闭锁的产品中的应用。
进一步地,所述检测CD177的物质包括检测CD177的试剂和/或仪器。
本发明还提供了CD177作为标志物在开发胆道闭锁的诊断产品中的应用。
进一步地,所述诊断产品包括试剂或试剂盒。
另外,本发明还提供了CD177的抑制剂在制备用于治疗胆道闭锁的产品中的应用。
进一步地,所述产品包括药物。
进一步地,所述CD177的抑制剂包括anti-mouse-Ly6G。
进一步地,所述CD177包括CD177基因、CD177蛋白或CD177+中性粒细胞。
相比于现有技术,本发明具有如下有益效果:
本发明通过检测大量的胆道闭锁患儿样本,并结合动物实验,首先证实了胆道闭锁外周血和肝脏组织中的CD177+中性粒细胞的数量可作为评估肝损伤程度的有力指标,因此检测CD177基因、CD177蛋白或CD177+中性粒细胞均可作为临床上评估胆道闭锁患儿的病情提供更方便、直接、准确的方法。对患儿检查年龄没有限制,能够保证诊断及治疗的时机。且检测特异性强,能够保证检测结果的准确性。
除此之外,本发明还证实了CD177在胆道闭锁中具有促炎的作用,其抑制剂可用于治疗胆道闭锁的药物研发。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1A为本发明实施例提供的使用流式细胞技术分析CD177+PMN在NC(n=8)、IHC(n=30)及BA(n=32)患儿外周血活细胞中的比例;
图1B为本发明实施例提供的使用流式细胞技术分析CD177+PMN在IHC(n=32)及BA(n=34)患儿肝脏组织活细胞中的比例;
图1C为本发明实施例提供的NC组、IHC组及BA组患儿肝脏CD177免疫组化染色代表性图片;
图1D为本发明实施例提供的肝脏中CD177+PMN比例与BA患儿术前外周血中γ-GT水平相关性分析;*p<0.05,**p<0.01,***p<0.001;NC:正常同年龄儿童;IHC:肝内胆汁淤积患儿;
图1E为本发明实施例提供的肝脏中CD177+PMN比例与BA患儿术前外周血中DBIL水平相关性分析;*p<0.05,**p<0.01,***p<0.001;NC:正常同年龄儿童;IHC:肝内胆汁淤积患儿;
图1F为本发明实施例提供的肝脏中CD177+PMN比例与BA患儿术前外周血中TBIL水平相关性分析;*p<0.05,**p<0.01,***p<0.001;NC:正常同年龄儿童;IHC:肝内胆汁淤积患儿;
图1G为本发明实施例提供的外周血中CD177+PMN比例与BA患儿术前外周血中γ-GT水平相关性分析;*p<0.05,**p<0.01,***p<0.001;NC:正常同年龄儿童;IHC:肝内胆汁淤积患儿;
图1H为本发明实施例提供的外周血中CD177+PMN比例与BA患儿术前外周血中DBIL水平相关性分析;*p<0.05,**p<0.01,***p<0.001;NC:正常同年龄儿童;IHC:肝内胆汁淤积患儿;
图1I为本发明实施例提供的外周血中CD177+PMN比例与BA患儿术前外周血中TBIL水平相关性分析;*p<0.05,**p<0.01,***p<0.001;NC:正常同年龄儿童;IHC:肝内胆汁淤积患儿;
图1J为本发明实施例提供的自第3天起,使用流式细胞技术分析CD177+PMN在Cont.(n=21)、BA(n=22)小鼠脾脏活细胞中的比例;*p<0.05,**p<0.01,***p<0.001;Cont.:正常Balb/c小鼠+生理盐水;RRV:正常Balb/c小鼠+RRV;
图1K为本发明实施例提供的自第3天起,使用流式细胞技术分析CD177+PMN在Cont.(n=21)、BA(n=22)小鼠肝脏活细胞中的比例;*p<0.05,**p<0.01,***p<0.001;Cont.:正常Balb/c小鼠+生理盐水;RRV:正常Balb/c小鼠+RRV;
图1L为本发明实施例提供的自第3天起,Cont.和BA组患儿肝脏CD177免疫组化染色代表性图片;*p<0.05,**p<0.01,***p<0.001;Cont.:正常Balb/c小鼠+生理盐水;RRV:正常Balb/c小鼠+RRV;
图2A为本发明实施例提供的第12天体内WT小鼠、RRV+WT小鼠、CD177–/–小鼠、RRV+CD177–/–小鼠外观代表性图片;
图2B为本发明实施例提供的1-21天各组小鼠体重曲线;
图2C为本发明实施例提供的1-21天各组小鼠生存曲线;
图2D为本发明实施例提供的第6、12天各组小鼠肝外胆管造影代表性图片;
图2E为本发明实施例提供的第6、12天各组小鼠肝内胆管染色代表性图片;
图2F为本发明实施例提供的第12天体内WT小鼠、RRV+WT小鼠、CD177–/–小鼠、RRV+CD177–/–小鼠肝脏外观代表性图片;
图2G为本发明实施例提供的第6、12天各组小鼠肝脏切片HE染色代表性图片;
图3A为本发明实施例提供的第1、2、3天人胆道上皮细胞对照组(BEC)、人胆道上皮细胞+正常健康儿童外周血中CD177+PMN共培养组(BEC+NC-CD177)、人胆道上皮细胞+BA患儿外周血中CD177+PMN共培养组(BEC+BA-CD177)细胞形态代表性图片;
图3B为本发明实施例提供的第三天各组胆道上皮细胞MTT细胞增殖实验统计结果。
具体实施方式
下面将结合实施例对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
除非本文另有定义,连同本发明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。术语的含义和范围应当清晰,然而,在任何潜在不明确性的情况下,本文提供的定义优先于任何字典或外来定义。在本申请中,除非另有说明,术语“包括”及其他形式的使用是非限制性的。
一般地,连同本文描述的细胞和组织培养、分子生物学、免疫学、微生物学、遗传学以及蛋白和核酸化学和杂交使用的命名法和其技术是本领域众所周知和通常使用的那些。除非另有说明,本发明的方法和技术一般根据本领域众所周知,且如各种一般和更具体的参考文献中所述的常规方法来进行,所述参考文献在本说明书自始至终引用和讨论。酶促反应和纯化技术根据制造商的说明书、如本领域通常实现的或如本文所述来进行。连同本文描述的分析化学、合成有机化学以及医学和药物化学使用的命名法、以及其实验室程序和技术是本领域众所周知和通常使用的那些。
CD177是属于Ly-6家族中的58-64-kDa蛋白,其通过糖基磷脂酰肌醇-键在嗜中性粒细胞中表达。在生理条件下,45%至60%中性粒细胞上表达CD177。
本发明通过实验发现,BA患儿肝脏中的CD177+中性粒细胞表达显著上调,且BA患儿中汇管区CD177+中性粒细胞与胆管在位置上是紧密相连的,胆道上皮细胞存在明显的损伤,提示CD177+中性粒细胞与BA的形成密切相关。本发明的前期预实验结果显示:(1)CD177+中性粒细胞在BA患儿肝脏和外周血及BA小鼠模型肝脏和脾脏中均显著增加,且与BA患儿直接胆红素、总胆红素和γ-GT呈正相关;(2)在动物实验中,本发明通过腹腔注射RRV感染CD177基因敲除新生小鼠,结果形成BA小鼠的数量及体重减低程度都大为减轻;(3)在细胞实验中,本发明分选BA患儿外周血中的CD177+中性粒细胞与人胆道上皮细胞(Biliaryepithelial cells,BEC)共培养,发现CD177+中性粒细胞可直接损伤BEC。以上实验结果均可表明,CD177+中性粒细胞在BA形成过程中起关键作用。
基于此,本发明提供了检测CD177的物质在制备用于诊断胆道闭锁的产品中的应用。以及,
检测CD177的物质在制备用于评估胆道闭锁患儿肝脏损伤程度的产品中的应用。以及,
检测CD177的物质在制备用于预后评估评估胆道闭锁的产品中的应用。
通过证实胆道闭锁外周血和肝脏组织中的CD177+中性粒细胞的数量可作为评估肝损伤程度的有力指标,本发明提出检测CD177基因、CD177蛋白或CD177+中性粒细胞均可作为临床上评估胆道闭锁患儿的病情提供更方便、直接、准确的方法。
需要进行说明的是,当检测得到的CD177基因、CD177蛋白或CD177+中性粒细胞的含量均高于(具有统计学差异,P<0.05)未患病的正常儿童时,则可诊断或辅助诊断为患有胆道闭锁;
对于已确诊胆道闭锁的患儿,当检测得到的CD177基因、CD177蛋白或CD177+中性粒细胞的含量越高时,则可诊断或辅助诊断为道闭锁患儿肝脏损伤程度越重,相应的,当检测得到的CD177基因、CD177蛋白或CD177+中性粒细胞的含量越低时,则可诊断或辅助诊断为道闭锁患儿肝脏损伤程度越轻;
对于进行了疾病干预后的胆道闭锁患儿,当检测得到的CD177基因、CD177蛋白或CD177+中性粒细胞的含量越高时,则可诊断或辅助诊断为胆道闭锁患儿的预后越差,相应的,当检测得到的CD177基因、CD177蛋白或CD177+中性粒细胞的含量越低时,则可诊断或辅助诊断为胆道闭锁患儿的预后越好。
此外,为了便于理解,在本发明中,CD177+中性粒细胞与CD177阳性中性粒细胞同意。
可选地,所述检测CD177的物质包括检测CD177的试剂和/或仪器。
例如,可以使用特异性引物检测CD177基因、使用特异性抗体检测CD177蛋白、使用流式细胞仪检测CD177+中性粒细胞等等。
根据本发明上述得出的CD177在BA形成过程中起关键作用的实验结果,本发明还提供了CD177作为标志物在开发胆道闭锁的诊断产品中的应用。
可选地,上述诊断产品可以为试剂或试剂盒。
此外,本发明还提供了CD177的抑制剂在制备用于治疗胆道闭锁的产品中的应用。
同样地,本发明还通过实验证实了CD177在胆道闭锁中具有促炎的作用,因此,其抑制剂可用于治疗胆道闭锁的药物研发。
可选地,所述产品可以为药物。
在一些优选的实施方式中,所述CD177的抑制剂包括抗体anti-mouse-Ly6G等。
最后需要说明的是,在本发明中,如无特殊说明,所述CD177可以为CD177基因、CD177蛋白或CD177+中性粒细胞中的一种或多种。除此之外,凡是能够量化CD177的其他物质也可作为本发明中所述的CD177,本发明对此不做限定。
下面通过具体的实施例进一步说明本发明,但是,应当理解为,这些实施例仅仅是用于更详细地说明之用,而不应理解为用于以任何形式限制本发明。
实施例1检测CD177+细胞在BA中的数量
(1)流式细胞术检测CD177+中性粒细胞
临床样本收集:1)BA及婴儿肝炎综合征患儿肝脏组织样本:经伦理审批和患者家属知情同意后,在胆道探查时或Kasai术中切取直径约1.5cm的肝右叶边缘新鲜组织样本;2)BA、婴儿肝炎综合征及同龄血管瘤患儿外周血样本:经伦理审批和患者家属知情同意后,收集术前且无抗生素干预的BA、婴儿肝炎综合征及同龄血管瘤患儿新鲜外周血样本;
小鼠肝脏组织样本收集:采用前述方法,建立BA小鼠模型并收集第3、6、9、12天的BA小鼠及正常小鼠肝脏和脾脏组织样本。单个核细胞分离:肝脏组织单个核细胞分离:分别将上述临床患儿肝脏组织样本及小鼠肝脏和脾脏组织样本分离单个核细胞,方法步骤详见前。
流式细胞分选:(人和小鼠)
(a)流式细胞分选人肝脏中CD177+细胞:收集BA患儿和CC患儿新鲜肝脏组织标本,将肝脏组织剪成约1mm3大小的组织块,使用美天妮公司的肝脏解离试剂盒将肝脏组织块解离成单细胞悬液,离心去上清收集细胞,加入5mL红细胞裂解液,室温裂解红细胞10min,使用流式染色缓冲液500g,5min离心洗涤细胞,调整所需细胞浓度,按照试剂说明书推荐用量加入Human Fc Block,室温孵育10min,按照流式抗体说明书操作依次加入anti-HumanCD45和anti-HumanCD177,4℃孵育30min,加入流式染色缓冲液500g、5min离心洗涤细胞,加入死细胞染料7-AAD,室温孵育5min,使用流式染色缓冲液调整所需细胞浓度,进行上机分选,使用BD公司FACSAria流式分选仪分选CD177阳性细胞;
(b)流式细胞分选小鼠肝脏中CD177阳性细胞:取小鼠新鲜肝脏标本,使用眼科剪将肝脏组织剪成约1mm3大小的组织块,使用美天妮公司的肝脏解离试剂盒将肝脏组织块解离成单细胞悬液,离心去上清收集细胞,加入5mL红细胞裂解液,室温裂解红细胞10min,使用流式染色缓冲液500g、5min离心洗涤细胞,调整所需细胞浓度,按照试剂说明书推荐用量加入Anti-Mouse CD16/CD32,室温孵育10min,按照流式抗体说明书操作依次加入anti-Mouse CD45和anti-MouseGr-1,4℃孵育30min,加入流式染色缓冲液500g、5min离心洗涤细胞,加入死细胞染料7-AAD,室温孵育5min,使用流式染色缓冲液调整所需细胞浓度,进行上机分选,使用BD公司FACSAria流式分选仪分选CD177阳性细胞。
(2)免疫组化检测肝脏组织CD177+细胞
通过免疫组化分别对BA、婴儿肝炎综合征和门脉海绵样变患儿及第3、6、9、12天BA和正常小鼠肝脏切片进行染色,并分析CD177+细胞的表达情况。抗体:Anti-HumanCD177(WH0057126M1,Sigma-Aldrich,USA)、Anti-MouseCD177(ab203025,ABCAM,UK)。详细染色方法见前期文章【ZhangRZetal.World Journal of Gastroenterology,2016;22(8):2545-2557.】。
本实施例使用流式细胞术和免疫组化染色检测分析了BA患儿肝脏和外周血中CD177+PMN的数量,结果显示:与正常对照儿童(NC组)及胆汁淤积患儿(IHC组)相比,BA组患儿外周血中CD177+PMN的数量显著增多(p<0.001,图1A)。与胆汁淤积患儿(IHC组)相比,BA组患儿肝脏中CD177+PMN的数量也显著增多(p<0.001,图1B),且大部分CD177+PMN分布在胆管周围(图1C)。同样的,在BA小鼠模型中,本实施例同样使用流式细胞术和免疫组化染色检测分析了BA小鼠肝脏和脾脏中CD177+PMN的数量,结果显示:与对照组小鼠相比,自第3天起BA组小鼠肝脏及脾脏中CD177+PMN均显著增多(p<0.001,图1J、图1K、图1L)。
(3)胆道闭锁患儿CD177+细胞表达水平与肝功指标的预后关联性分析采集BA患儿的术前、术后的临床病例资料,检测血样和肝脏组织的CD177+细胞表达水平,与临床肝功指标(包括直接胆红素(DBIL)、总胆红素(TBIL)、γ-GT等)进行关联性分析及临床预后的回顾性分析。
为探索CD177+PMN的数量与胆汁淤积的关系,本实施例分析了BA患儿肝脏和外周血中CD177+PMN比例与BA患儿术前外周血中γ-GT、DBIL及TBIL水平的相关性。本实施例观察到BA患儿肝脏和外周血中CD177+PMN比例均分别与BA患儿术前外周血中γ-GT(R=0.63,P<0.0001;R=0.41,P=0.0339)、DBIL(R=0.73,P<0.0001;R=0.67,P<0.0001)及TBIL(R=0.57,P=0.0004;R=0.61,P=0.0005)水平呈正相关(如图1D、1E、1F、1G、1H和1I所示)。上述结果提示CD177+PMN在BA的发病过程中起重要作用。
实施例2在基因敲除小鼠上进一步证实CD177+细胞在BA中的重要性
CD177基因敲除小鼠感染RRV实验:
(1)分别在前期培育的CD177基因敲除小鼠(CyagenQuote:KOCMS180821DA4+KOCMS190314DA2-B)出生24h内腹腔注射RRV20μL(滴度1.5×106PFU)。
(2)将小鼠随机分为:①仅注射RRV组;②正常小鼠对照组。对照组于同时间段注射等量生理盐水。收取以上分组的第3、6、9、12天小鼠肝脏组织样本。
(3)观察小鼠的体重、皮肤黄疸、生存率及肝功能的变化;常规胆囊注射美蓝进行肝外胆道造影,确认是否存在肝外胆道闭锁;病理染色观察小鼠肝内、外胆管形态变化。
为进一步明确CD177+PMN在BA中的作用,本实施例建立起CD177–/–Balb/c小鼠模型,并按照BA小鼠的造模方法观察小鼠的变化。结果显示,在RRV感染后的第六天起,与感染RRV的WT小鼠相比,感染RRV的CD177–/–小鼠黄疸率显著降低,体重降低程度减轻,生存率显著提高,生存时间显著延长(图2A、图2B、图2C),肝脏表面未见点状炎症坏死灶(图2F),肝外胆管未发生闭锁(图2D),肝内胆管周围未见明显的炎症细胞浸润(图2G),肝内胆管也未发生闭锁(图2E)。上述结果表明CD177+PMN在小鼠BA的发病过程中起至关重要的作用。
实施例3体外实验探索CD177+细胞对胆道上皮细胞(BEC)的影响
1、CD177+细胞与正常胎儿BEC体外共培养:
(1)BA患儿的CD177+细胞分选:收集BA、婴儿肝炎综合征及同龄血管瘤患儿外周血样本,通过流式细胞分选方法分选出外周血中的CD177+细胞(详细步骤见实施例1)。
(2)分选细胞与正常胎儿BEC体外共培养:将人胚胎胆管上皮细胞(BEC)铺在经过Poly-L-Lysine(PLL)包被的T75培养瓶中,置于37℃,5%CO2培养箱中培养扩增,其中胆道上皮细胞购买于ScienCell公司。将上述步骤提取的BA患儿及疾病对照组的肝脏或血样的CD177+细胞加入BEC培养基中进行重悬,至细胞数为1×105个/mL,镜下分别对24h、48h、72h的BEC形态结构进行拍照。
(3)流式凋亡染色方法检测BEC凋亡水平:使用eBioscienceAnnexin VApoptosisDetectionKitPE对其进行孵育染色,用流式细胞仪(BDFACSCantoTM IIsystem)上机检测BEC凋亡情况。
2、小鼠CD177+细胞与小鼠BEC体外共培养:
(1)分选BA小鼠的CD177阳性细胞及CD177阴性细胞:收集第3、6、9、12天BA小鼠新鲜肝脏组织样本,通过流式细胞分选方法分选出BA小鼠肝脏中的CD177阳性细胞及CD177阴性细胞(详细步骤见上文)。
(2)共培养及检测同上。
为进一步探讨CD177+PMN在BA发病中的作用及其机制,本实施例分别从BA患儿和同龄正常健康儿童外周血中分选出CD177+PMN,体外与人胆道上皮细胞(BEC)共培养。结果显示,从共培养的第二天起,与同龄正常健康儿童的CD177+PMN共培养的BEC细胞相比,与BA患儿的CD177+PMN共培养的BEC细胞形态异常,生长缓慢,第三天各组MTT细胞增殖实验结果显示,与BA患儿的CD177+PMN共培养的BEC细胞增殖受到显著抑制(图3A、3B),表明BA患儿的CD177+PMN可导致人BEC细胞损伤。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (4)
1.定量检测CD177的物质在制备用于诊断胆道闭锁的产品中的应用;所述CD177来源于外周血;所述CD177为CD177+中性粒细胞。
2.定量检测CD177的物质在制备用于评估胆道闭锁患儿肝脏损伤程度的产品中的应用;所述CD177来源于外周血;所述CD177为CD177+中性粒细胞。
3.定量检测CD177的物质在制备用于预后评估胆道闭锁的产品中的应用;所述CD177来源于外周血;所述CD177为CD177+中性粒细胞。
4.根据权利要求1-3任一项所述的应用,其特征在于,所述检测CD177的物质包括检测CD177的试剂和/或仪器。
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