CN113655142B - 一种基于磷脂酰丝氨酸和磷脂酰乙醇胺的早期预警重症急性胰腺炎的模型与应用 - Google Patents
一种基于磷脂酰丝氨酸和磷脂酰乙醇胺的早期预警重症急性胰腺炎的模型与应用 Download PDFInfo
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Abstract
本发明公开了一种基于磷脂酰丝氨酸和磷脂酰乙醇胺的早期预警重症急性胰腺炎的模型与应用,属于生物检测技术领域。所述预测模型为血清中磷脂酰丝氨酸(38:3)和磷脂酰乙醇胺(16:0/20:3),通过检测血清中磷脂酰丝氨酸(38:3)和磷脂酰乙醇胺(16:0/20:3)的含量变化,在患者患病48h内,区分中重症急性胰腺炎和重症急性胰腺炎,具有良好的临床意义和应用价值。
Description
技术领域
本发明涉及一种基于磷脂酰丝氨酸和磷脂酰乙醇胺的早期预警重症急性胰腺炎的模型与应用,属于生物检测技术领域。
背景技术
脂质组学(lipidomics)是一种基于液相色谱-质谱联用(LC-MS/MS)高通量分析技术,系统性解析生物体脂质组成与表达变化的研究模式,可有效研究脂类家族、脂质分子在各种生物过程中的改变与功能,进而阐明相关的生命活动机制与机理。液质联用技术策略是利用不同的脂质提取方法分别提取不同种类的脂质,或根据不同脂质种类的极性差异,利用正相色谱在种类的水平上将生物样本的脂质分为不同的组分,然后利用反相色谱将组分中的脂质分子(molecular species)进一步分离,进而利用质谱进行定性、定量分析。
急性胰腺炎是常见的消化系统疾病之一,其特点是发病快、进展迅速,并且发病率逐年增高。早期有效的液体复苏与合理的支持治疗,是目前主要的治疗策略。2012年最新亚特兰大分类根据急性胰腺炎病情严重程度,将急性胰腺炎分为轻症急性胰腺炎(MAP)、中度重症急性胰腺炎(MSAP)、重症急性胰腺炎(SAP)。轻度急性胰腺炎是一种自限性疾病,经对症治疗后患者预后较好。值得注意的是,约20%的患者会进展为重症急性胰腺炎,死亡率高达10%-30%,即使幸存的患者也伴有严重的近期与远期并发症。很大部分原因在于目前尚无精确反应胰腺炎严重程度的血液标志物或评分量表,延误了治疗时间窗。重症急性胰腺炎的早期预警仍然是临床诊疗过程中的一项重大挑战。因此,需要进一步的发掘具有高诊断效能标志物,对具有重症转化风险的患者早期预警,选择合适的治疗策略,具有重要的临床意义。
发明内容
磷脂酰丝氨酸(PS)和磷脂酰乙醇胺(PE)均为细胞中重要的膜磷脂,维持细胞的稳态。本发明基于液质联用的脂质组学技术对血清磷脂酰丝氨酸PS(38:3)和磷脂酰乙醇胺PE(16:0/20:3)实现精确定性和精准定量,以达到早期预警重症急性胰腺炎(SAP)的目的。
一种基于磷脂酰丝氨酸和磷脂酰乙醇胺的早期预警重症急性胰腺炎的预测模型,所述预测模型为磷脂酰丝氨酸(38:3)和磷脂酰乙醇胺(16:0/20:3),所述磷脂酰丝氨酸(38:3)指有38个碳原子不饱和度为3的磷脂酰丝氨酸,所述磷脂酰乙醇胺(16:0/20:3)指两条脂肪酸链分别有16个碳原子完全饱和,20个碳原子不饱和度为3的磷脂酰乙醇胺,通过检测血清中磷脂酰丝氨酸(38:3)和磷脂酰乙醇胺(16:0/20:3)的含量变化,在患者患病48h内,区分中重症急性胰腺炎和重症急性胰腺炎。
进一步地,上述技术方案中,所述检测血清中磷脂酰丝氨酸(38:3)和磷脂酰乙醇胺(16:0/20:3)的含量变化为:
相较于健康人,重症急性胰腺炎患者血清中磷脂酰丝氨酸(38:3)表达上调>3.5倍,磷脂酰乙醇胺(16:0/20:3)表达上调>4.2倍;
相较于健康人,中重症急性胰腺炎患者血清中磷脂酰丝氨酸(38:3)表达上调1.5~3.5倍,磷脂酰乙醇胺(16:0/20:3)表达上调1.7~4.2倍。
进一步地,上述技术方案中,血清预处理,提取脂质,通过液相色谱分离,再利用质谱仪定量分析,检测血清中磷脂酰丝氨酸(38:3)和磷脂酰乙醇胺(16:0/20:3)的含量,在患者患病起48h内,区分中重症急性胰腺炎和重症急性胰腺炎。
进一步地,上述技术方案中,血清预处理的方法包括:
(1)向血清中加入甲醇,涡旋震荡;
(2)加入甲基叔丁基醚,用于提取脂质,再加入超纯水再次涡旋振荡;
(3)室温下摇匀后,静置分层,离心;
(4)将上层脂质提取物减压干燥,密封低温保存;
(5)加入乙腈-异丙醇溶液复溶,涡旋震荡,离心;
(6)取上清液进行液相色谱-质谱联用检测。
进一步地,上述技术方案中,血清:甲醇:甲基叔丁基醚的体积比为1:5~7:16~20。
进一步地,上述技术方案中,乙腈:异丙醇的体积比为1:0.8~1:1.2。
进一步地,上述技术方案中,使用液相色谱和质谱时,所用的试剂均为色谱级,包括:甲醇、乙腈、异丙醇、甲酸、甲酸铵和甲基叔丁基醚。
进一步地,上述技术方案中,液相色谱的流动相A为:55%~65%乙腈-水,流动相B为8%~12%乙腈-异丙醇,流动相A和流动相B中均含有8~12mmol/L甲酸铵和0.08~0.12%甲酸;分离梯度为:初始浓度为8%~12%的B相在4~6分钟内上升到50%,在20~25分钟内进一步上升到100%。
一种早期预警重症急性胰腺炎的预测模型的应用,用于48h内区分中重症急性胰腺炎和重症急性胰腺炎。
发明有益效果
通过本发明所述方法,能够相对快速、精准的定量血清(血浆)中磷脂分子:磷脂酰丝氨酸PS(38:3)和磷脂酰乙醇胺PE(16:0/20:3),可重复性好。
本发明所述急性胰腺炎(AP)严重程度分级是根据2012急性胰腺炎亚特兰大分类标准,但该分类标准对于中重症和重症的区分存在48h的等待时间窗:即AP患者伴有器官衰竭,48h内恢复即为中重症,48h内未恢复即为重症。而本发明的联合测定磷脂酰丝氨酸PS(38:3)和磷脂酰乙醇胺PE(16:0/20:3)能够在相对早期(48h内)辅助区分中重症和重症(见ROC曲线结果),具有良好的指导意义。
附图说明
图1为磷脂酰丝氨酸PS(38:3)的液相色谱图(A)和质谱图(B)。
图2为磷脂酰乙醇胺PE(16:0/20:3)的液相色谱图(A)和质谱图(B)。
图3为磷脂酰丝氨酸PS(38:3)在不同组的丰度。
图4为磷脂酰乙醇胺PE(16:0/20:3)在不同组的丰度。
图5为磷脂酰丝氨酸PS(38:3)和磷脂酰乙醇胺PE(16:0/20:3)的ROC曲线。
具体实施方式
下述非限定性实施例可以使本领域的普通技术人员更全面地理解本发明,但不以任何方式限制本发明。
实施例1
脂质组学研究的技术主要包括脂质的提取、分离以及检测。
试剂:色谱级甲醇、乙腈、异丙醇、甲酸和甲酸铵购自Fisher Science(Fair Lawn,USA)。色谱级甲基叔丁基醚(MTBE)购自Sigma-Aldrich(St.Louis,USA)。超纯水采用MILI-Q纯净水系统制备(Merck KGaA,Darmstadt,Germany)。
仪器:Ultal3000型超高效液相色谱仪和QExactive高分辨质谱仪(ThermoScientific,USA)组成。
样本前处理:
1.取20μL患者血清至1.5mL EP管中,精确加入120μL甲醇溶液,涡旋震荡1500rpm,3min。
2.加入360μL色谱级甲基叔丁基醚(MTBE)(用于提取脂质),再加入100μL超纯水再次涡旋振荡1500rpm,3min。
3.室温下摇匀10min后,4℃静置10min以促进分层,随后4℃下13000g离心10min。
4.定量转移上层脂质提取物200μL至新EP管中减压干燥,密封低温保存。
5.加入100μL乙腈-异丙醇溶液(1:1,v/v)复溶,涡旋震荡3分钟,4℃条件下13000g离心10分钟。
6.取上清液90μL上样检测。
脂类检测:包括色谱分离和质谱检测
色谱分离:采用Accucore C30 core-shell色谱柱(Thermo Scientific,USA,2.6μm,2.1×100mm)。60%乙腈-水为A相,10%乙腈-异丙醇为B相,二者均含有10mmol/L甲酸铵和0.1%甲酸。分离梯度为:初始浓度为10%的B相在5分钟内上升到50%,在23分钟内进一步上升到100%。其余7分钟用于柱子洗涤和平衡。流速、进样量和柱温分别为0.3mL/min、2μL和50℃。
质谱检测:使用加热的电喷雾电离源(ESI+),各项参数如下:鞘气45arb;辅助气体10arb;加热器温度355℃;毛细管温度320℃;S-Lens RF level 55%。代谢组提取物在全扫描模式(FS)下进行,分辨率为70000FWHM,最大进样时间为200ms,自动增益控制目标(AGC)为3×106。数据采集使用300~2000m/z的扫描范围。
利用该检测方法分别对102例受试者血清样本中磷脂酰丝氨酸PS(38:3)和磷脂酰乙醇胺PE(16:0/20:3)进行检测,包括轻度(39例)、中重症(20例)、重症(19例)急性胰腺炎患者和健康对照者(24例)。结果显示:PS和PE均随着疾病严重程度的增加而上升(如图3、图4);且二者对SAP具有较好的区分效力,PS(38:3)的AUC值为0.9,特异性和敏感性均为82.4%;PE的AUC值为0.817,特异性和敏感性分别为70.6%,82.4%。两者结合的AUC值为0.9,特异性和敏感性分别为82.4%,二者结合可早期(48h内)区分中重症急性胰腺炎和重症急性胰腺炎(如图5)。
Claims (7)
1.磷脂酰丝氨酸和磷脂酰乙醇胺在制备早期预警重症急性胰腺炎试剂盒中的应用,其特征在于,所述磷脂酰丝氨酸为磷脂酰丝氨酸38:3,指有38个碳原子不饱和度为3的磷脂酰丝氨酸,所述磷脂酰乙醇胺为磷脂酰乙醇胺16:0/20:3,指两条脂肪酸链分别有16个碳原子完全饱和,20个碳原子不饱和度为3的磷脂酰乙醇胺;通过检测血清中磷脂酰丝氨酸和磷脂酰乙醇胺的含量变化,在患者患病48h内,区分中重症急性胰腺炎和重症急性胰腺炎;
所述检测血清中磷脂酰丝氨酸和磷脂酰乙醇胺的含量为:
相较于健康人,重症急性胰腺炎患者血清中磷脂酰丝氨酸表达上调> 3.5倍,磷脂酰乙醇胺表达上调>4.2倍;
相较于健康人,中重症急性胰腺炎患者血清中磷脂酰丝氨酸表达上调1.5~3.5倍,磷脂酰乙醇胺表达上调1.7~4.2倍。
2.根据权利要求1所述的应用,其特征在于,检测血清中磷脂酰丝氨酸和磷脂酰乙醇胺含量的方法为:血清预处理,提取脂质,通过液相色谱分离,再利用质谱仪定量分析,检测血清中磷脂酰丝氨酸和磷脂酰乙醇胺的含量,在患者患病起48h内,区分中重症急性胰腺炎和重症急性胰腺炎。
3.根据权利要求2所述的应用,其特征在于,血清预处理的方法包括:
(1)向血清中加入甲醇,涡旋震荡;
(2)加入甲基叔丁基醚,用于提取脂质,再加入超纯水再次涡旋振荡;
(3)室温下摇匀后,静置分层,离心;
(4)将上层脂质提取物减压干燥,密封低温保存;
(5)加入乙腈-异丙醇溶液复溶,涡旋震荡,离心;
(6)取上清液进行液相色谱-质谱联用检测。
4.根据权利要求3所述的应用,其特征在于,血清:甲醇:甲基叔丁基醚的体积比为1:5~7:16~20。
5.根据权利要求4所述的应用,其特征在于,乙腈:异丙醇的体积比为1:0.8~1:1.2。
6.根据权利要求2所述的应用,其特征在于,使用液相色谱和质谱时,所用的试剂均为色谱级,包括:甲醇、乙腈、异丙醇、甲酸、甲酸铵和甲基叔丁基醚。
7.根据权利要求2所述的应用,其特征在于,液相色谱的流动相A为:55%~65%乙腈-水,流动相B为8%~12%乙腈-异丙醇,流动相A和流动相B中均含有8~12mmol/L甲酸铵和0.08~0 .12%甲酸;分离梯度为:初始浓度为8%~12%的B相在4~6分钟内上升到50%,在20~25分钟内进一步上升到100%。
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