CN110068682A - 血小板微颗粒在细胞组织因子表达升高中的用途 - Google Patents
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Abstract
本发明血小板微颗粒在白血病细胞组织因子表达升高并发症中的用途,属于生物医学领域。具体地,本发明公开了血小板微颗粒在制备用于诊断或预测白血病患者因细胞组织因子表达水平升高的试剂中用途,以及膜联蛋白V在制备或筛选用于预防或治疗白血病患者因细胞组织因子表达水平升高引起的并发症的药物中用途。本发明应用于临床将对恶性肿瘤特别是血液肿瘤患者危急状况的评估和及时救治有重要价值。
Description
技术领域
本发明属于生物医学领域,具体地,涉及一种血小板微颗粒在细胞组织因子表达升高中的用途。
背景技术
细胞组织因子(cTF)正常处在封闭状态,而细胞组织因子的激活被众多学者称之为cTF“解密”。
血小板微颗粒(platelet microparticle,PMP)是血小板在不同刺激因素作用下细胞膜向外伸展变形的细胞膜部分以出芽方式形成小泡或伪足脱落进入微循环而形成细小微粒,是血小板活化的标志物之一。其直径小于0.5μm,不能用普通显微镜观察到其形态,不能用包括血小板计数仪在内的常规检测血小板的方法来检测。PMP体积小于正常的血小板但具有完整的膜结构,血小板微粒膜上携带有静息状态下血小板膜上的多数成分,也携带活化血小板膜上的标记物(如CD62p等)。它具有很强的促凝活性,也有一定的抗凝活性,在循环血中PMP增高与许多临床疾病相关,在人体血栓与止血过程中发挥重要作用。
很早以前人们就认识到血小板在凝血过程中的作用,因为乏血小板血浆的凝固时间要比富血小板血浆长很多。但是,人们发现,往将PMP加入抗凝全血中可大大缩短其凝固时间;往血浆中加入PMP,其凝固时间无明显变化。1967年,Wolf首先发现并描述了血小板微粒以及其促凝血活性,但对细胞组织因子的“解密”尚未做研究。
急性白血病(AL)凝血、纤溶功能的异常已有诸多报道,特别是AL伴发致死性血栓形成和出血已受到广泛的关注。由于血管损害、白血病细胞的侵润和重度感染等病理因素导致的急性弥散性血管内凝血(DIC)往往是AL患者死亡的主要原因之一。因此,临床急需防止白血病出血和血栓形成并发症和提升有效抢救率的新途径。
发明内容
为了解决上述技术问题,发明人通过采用简便易行、快捷敏感的技术检测不同类型白血病细胞的组织因子表达的激活,出乎意料地发现经血小板微颗粒(PMP)作用的非淋巴细胞白血病细胞具有较正常白细胞明显增强的组织因子表达,并且不同类型的白血病细胞TF的表达存在差异,从而完成本发明。
本发明的第一方面提供血小板微颗粒在制备用于诊断或预测白血病患者细胞组织因子表达水平升高的试剂中用途。
在本发明的一些实施方案中,所述试剂包括血小板微颗粒标记试剂。
在本发明的一些具体实施方案中,所述血小板微颗粒标记试剂为特异性抗体。
在本发明中,所述血小板微颗粒是通过下述方法制备的:
(1)富含血小板血浆(PRP)制备:抽取清晨空腹静息状态下静脉血,前2 mL血弃去,加入109 mmol/L枸橼酸钠(1:9)抗凝管,其中有10例健康体检者抽取2.7 mL于枸橼酸钠抗凝管,另外2.7 mL于CTAD抗凝管。混匀以后立即离心,1100 rpm/min离心10分钟;取上层富含血小板血浆;调整PRP血小板数为3×1011/L。
(2)血小板活化:200 μL PRP中加入激活剂ADP(终浓度为20μmol/L),胶原(终浓度为20 μg/L),室温孵育10min,孵育过程中避免摇动。
(3)免疫荧光标记:取10μL未加激活剂或加激活剂活化的PRP加入5μL FITC-CD61室温避光孵育30 min,加入1%冷多聚甲醛1mL终止反应,同时固定标本,2小时之内上机检测。待测标本上机前均加入用PBS做1:400稀释的标准微球1μL。
在本发明的一些实施方案中,所述试剂还包括血小板微颗粒分离试剂。
本发明的第二方面提供一种用于诊断或预测白血病患者细胞组织因子表达水平升高的试剂盒,所述试剂盒包括血小板微颗粒标记试剂。
在本发明的一些具体实施方案中,所述血小板微颗粒标记试剂为特异性抗体。
在本发明的一些实施方案中,所述试剂盒中还包括血小板微颗粒分离试剂。
本发明的第三方面提供膜联蛋白V(Annexin V)在制备用于预防或治疗白血病患者因细胞组织因子表达水平升高而引起的并发症的药物中用途。
在本发明的一些实施方案中,所述膜联蛋白V能够抑制血小板微颗粒激活白血病细胞组织因子。
本发明中,发明人通过特异性检测膜联蛋白V,证实抑制磷脂酰丝氨酸的作用可以有效抑制PMP刺激白血病细胞TF激活。
在本发明中,所述并发症为血栓形成和凝血成分消耗性出血。
在本发明的中,所述白血病为非淋巴细胞白血病。
本发明的有益效果
本发明利用TF的表达病理机制并有较高的特异性,具有简捷、快速的特点,应用于临床将对恶性肿瘤特别是血液肿瘤患者危急状况的评估和及时救治有重要价值。
附图说明
图1示出了TF-mRNA的检测结果,M:maker,1-2:PMPs“解密”刺激后淋巴细胞,3-4:PMPs“解密”后白血病细胞。
图2示出了“解密“前(A)后(B)白血病细胞c-TF表达(流式细胞仪检测)。
图3示出了两组表达荧光的细胞百分比和“解密”后细胞相对平均荧光表达率。
具体实施方式
为了使本发明所解决的技术问题、技术方案及有益效果更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。
实施例
以下例子在此用于示范本发明的优选实施方案。本领域内的技术人员会明白,下述例子中披露的技术代表发明人发现的可以用于实施本发明的技术,因此可以视为实施本发明的优选方案。但是本领域内的技术人员根据本说明书应该明白,这里所公开的特定实施例可以做很多修改,仍然能得到相同的或者类似的结果,而非背离本发明的精神或范围。
除非另有定义,所有在此使用的技术和科学的术语,和本发明所属领域内的技术人员所通常理解的意思相同,在此公开引用及他们引用的材料都将以引用的方式被并入。
那些本领域内的技术人员将意识到或者通过常规试验就能了解许多这里所描述的发明的特定实施方案的许多等同技术。这些等同将被包含在权利要求书中。
实施例 1 实验方案
1、PMP的制备
(1)富含血小板血浆(PRP)制备:抽取清晨空腹静息状态下静脉血,前2 mL血弃去,加入109 mmol/L枸橼酸钠(1:9)抗凝管,其中有10例健康体检者抽取2.7 mL于枸橼酸钠抗凝管,另外2.7 mL于CTAD抗凝管。混匀以后立即离心,1100 rpm/min离心10分钟;取上层富含血小板血浆;调整PRP血小板数为3×1011/L。
(2)血小板活化:200 μL PRP中加入激活剂ADP(终浓度为20μmol/L),胶原(终浓度为20 μg/L),室温孵育10min,孵育过程中避免摇动。
(3)免疫荧光标记:取10μL未加激活剂或加激活剂活化的PRP加入5μL FITC-CD61室温避光孵育30 min,加入1%冷多聚甲醛1mL终止反应,同时固定标本,2小时之内上机检测。待测标本上机前均加入用PBS做1:400稀释的标准微球1μL。
2、PMP的检定
(1)流式细胞仪测定
标本检测在Becton-Dickinson流式细胞仪上进行,以Flow check校准仪器光路;建立前向角光散射对侧向角光散射对数散点图(FSC LOG-SSC LOG):根据微球作为定位对照,调整机器的阈值,使每管均在同一检测条件及程序设定下检测;在CD61-FSC对数散点图上根据标准微球定位设门计数血小板微颗粒占CD61阳性颗粒的相对百分率。
(2)数据统计及分析
实验数据以X±s表示,应用SPSS软件进行t检验,方差分析,结果见表1。
表1 血小板活化前后释放的PMP量
※表示差异显著(P<0.05)。
上述结果表明,激活后PMPs数量明显增多。
2、单核、白血病细胞的的分离、培养、诱导培养和检定
细胞的培养与诱导:用含有10%胎牛血清的RPMI1640培养液于37℃,5%CO2恒温培养箱中培养细胞。台盼蓝拒染测细胞存活量为1×106个/mL。两组分别加制备好的PMPs,另一组设为阴性对照(加PBS)。
3、RT-PCR检测TF mRNA
分别收集两组细胞:
(1)加入PMPs悬液30μL于37℃孵育“解密”4h后的细胞液;
(2)未加任何试剂的同样条件下放置的未“解密”的细胞液100μL。
(3)用Biozol分别提取两组“解密”和未“解密”细胞总RNA。
(4)取两组细胞的总RNA各5μL,加随机引物0.5μL,于70℃水浴2分钟以上。用ddH2O、dNTP、RNA酶抑制剂、5×buffer、冰浴逆转录酶配成RT液。将RT液20μL/管加入70℃后的管中,42℃逆转录1h,得到cDNA。取产物2.5μL加入PCR反应液进行PCR循环(首先94℃预变性4 min;94℃变性30sec,57℃退火40sec,72℃延伸40sec,共30个循环;然后72℃延伸5min,4℃储存)。PCR反应液包括ddH2O、dNTP、10×buffer、Mg2+,加入冰浴Taq酶和引物。反应后取终产物6μL在1.0%的琼脂糖凝胶中电泳分析并拍照,目测目的基因及GAPDH条带,观察两组mRNA的相对表达量。
4、流式细胞术(FCM)检测“解密”细胞的TF表达
用含5%胎牛血清的RPMI1640培养液调整细胞密度至1×106个/mL,于4个流式管中分别加入100μL的细胞液。向③、④管中分别加入30μL分离的PMPs液和10μL浓度为125μg/mL的LPS后,同时将4管于37℃孵育4h。每管细胞用冷PBS洗涤后,用2mL的1%冷多聚甲醛避光固定30min,再用冷PBS洗涤。然后向②、③、④流式管的20μL密度为1×106个/mL ECV-304细胞液中分别加入5μL TF-FITC,①对应管中加入等量鼠IgG1-FITC作为阴性同型对照。将4管细胞液避光冰浴30min,冷PBS洗涤,1%冷多聚甲醛重悬后,上机检测。
5、RT-PCR检测cTF-mRNA
结果显示PMP刺激后单核细胞TF-mRNA表达量明显较刺激前增高(表1、图1),淋巴细胞刺激后未见有TF-mRNA表达。
实施例 2 “解密”细胞组织因子的流式细胞术检测
1、白血病细胞加入适量PMP悬液,在恒温(37℃)条件下进行充分激发,FCM结果显示(图2),未“解密”组0.91%细胞表达TF蛋白,细胞平均荧光表达率为1.52%;PMPs“解密”组32.97%细胞表达TF蛋白,细胞平均荧光表达率为32.26%;细胞孵育4h可诱导较多的细胞表达TF蛋白,且单个细胞所表达的TF蛋白量也明显增多。
2、激发后白血病细胞促凝固活性和TF表达量的检定
两组表达荧光的细胞百分比和“解密”后细胞相对平均荧光表达率如图3所示。
3、各型白血病患者cTF
解密后”促凝血活性(cTF-PCA)检查结果见表2。
表2 各型白血病患者TF-PCA测定结果( ±s)
表2结果表明,急性非淋巴细胞白血病(ANLL)组I、急性非淋巴细胞白血病组II,TF促凝活性较急性淋巴细胞(ALL)组,白血病细胞显著增强,验证c-TF表达增强。
4、临床跟踪
资料显示,ANLL组I、ANLL组II伴发急性致命性出血患者TF-PCA增强者DIC发生率为21.5%。
5、血小板微颗粒、乳糜微粒、P-选择素、细胞坏死因子“解密”后促凝活性:
取ANLL细胞用上述刺激因子刺激后,检测各自促凝活性,结果见表3。
表3 各种刺激因子促凝活性比较
表3结果提示PMP“解密”刺激白细胞后有较强的促凝血活性,与刺激前差异有极显著意义,细胞坏死因子有较弱的促凝血活性,乳糜微粒、P-选择素则没有直接激活c-TF的作用,同时说明PMP是最有效的“秘钥”。
以上结果表明,血液肿瘤细胞在适量血小板微颗粒(PMPs)和适当温度(37℃)的作用下,cTF表达增强,与血液中凝血因子VIIa形成复合物,同时激活凝血酶原转变为凝血酶,反馈激活血小板形成凝血激活循环,导致血栓形成和凝血成分消耗性出血。
由该结果可知,利用血小板微颗粒标记试剂检测白血病患者血浆中的血小板微颗粒含量,将能够诊断或预测白血病患者因细胞组织因子表达水平升高引起的并发症,如血栓形成和凝血成分消耗性出血。
发明人进一步通过建立大鼠动脉粥样硬化动物模型,采用细胞培养和PCR技术研究活体条件下白血病细胞中TF的激活释放及膜联蛋白V(Annexin V)的抑制作用。
结果表明,膜联蛋白V可以通过抑制磷脂酰丝氨酸的作用,显著抑制PMP刺激白血病细胞组织因子的激活,从而有效预防和治疗白血病患者因细胞组织因子表达水平升高引起的并发症,如血栓形成和凝血成分消耗性出血。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (10)
1.血小板微颗粒在制备用于诊断或预测白血病患者细胞组织因子表达水平升高的试剂中用途。
2.根据权利要求1所述的用途,其特征在于,所述试剂包括血小板微颗粒标记试剂。
3.根据权利要求2所述的用途,其特征在于,所述试剂还包括血小板微颗粒分离试剂。
4.一种用于诊断或预测白血病患者细胞组织因子表达水平升高的试剂盒,其特征在于,所述试剂盒包括血小板微颗粒标记试剂。
5.根据权利要求4所述的试剂盒,其特征在于,所述试剂盒中还包括血小板微颗粒分离试剂。
6.根据权利要求2所述的用途或权利要求4所述的试剂盒,其特征在于,所述血小板微颗粒标记试剂为特异性抗体。
7.膜联蛋白V在制备用于预防或治疗白血病患者因细胞组织因子表达水平升高而引起的并发症的药物中用途。
8.根据权利要求7所述的用途,其特征在于,所述膜联蛋白V能够抑制血小板微颗粒激活白血病细胞组织因子。
9.根据权利要求7或8所述的用途,其特征在于,所述并发症为血栓形成和凝血成分消耗性出血。
10.根据权利要求1所述的用途、权利要求4所述的试剂盒或权利要求7所述的用途,其特征在于,所述白血病为非淋巴细胞白血病。
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