CN111337684A - 一种肝纤维化抗体蛋白芯片的制备以及在诊断纤维化分期中的应用 - Google Patents
一种肝纤维化抗体蛋白芯片的制备以及在诊断纤维化分期中的应用 Download PDFInfo
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Abstract
本发明公开了肝纤维化抗体蛋白芯片的制备以及在诊断纤维化分期中的应用,包括不同病因肝纤维化患者血清生物标志物的样本库建立、敏感度和特异度较高的生物标志物筛选、肝组织活检分期的抗体蛋白芯片制备以及肝纤维化标志物蛋白抗体芯片检测系统、应用于医学研究和临床医学领域。采用本发明只需采取患者的外周血就可以快速高效的检测出肝纤维化的病因,降低了检测成本并且缩短了检测时间,减少了肝脏病变活检的需要,降低了取样难度并且减轻了因取样给患者带来的痛苦。
Description
技术领域
本发明属于生物技术领域,特别是涉及一种肝纤维化抗体蛋白芯片的制备以及在诊断纤维化分期中的应用。
背景技术
肝纤维化是多种慢性肝病共同归宿,活检是公认的临床诊断“金标准”。但由于其有创性以及相关并发症,血清学标志物的检测更具吸引力。目前已发现多种肝纤维化的生物标志物以及预测模型,然而各种单一的生物标志物和联合评分系统对不同病因导致的纤维化有偏向,同时敏感度和特异度各有优劣,为临床工作增加困难。因此,建立不同病因肝纤维化患者血液生物标志物的样本库,筛选出敏感度特异度较高的生物标志物,制备具有商业化潜能的抗体蛋白芯片,并能对应肝组织活检分期,将减少患者痛苦,更好地指导临床医师合理诊治肝纤维化,同时为肝纤维化的研究提供更多数据与资源。
肝纤维化诊断及治疗共识(2019年)共识与指南中推荐临床上肝纤维化的病理学诊断采用Scheuer评分系统:Scheuer评分系统将肝纤维化分为0~4期。0期-无纤维化;1期-门管区扩大;2期-门管区纤维化,纤维间隔形成;3期-纤维间隔伴小叶结构紊乱;4期-可能或肯定肝硬化
抗体芯片技术作为一种通量化的蛋白质分析技术,已经用于疾病蛋白质组的研究,并能够较全面和准确地反映出在疾病发生发展过程中的蛋白质表达水平的变化。
目前无大规模不同病因肝纤维化患者血清样本库,尚缺乏血清特异性肝纤维化诊断指标,同时单一血液指标对肝纤维化评估作用有限。并且无血清标志物模型对应肝组织活检分期。
发明内容
本发明的目的在于建立不同病因肝纤维化患者血清生物标志物的样本库,其特点在于能够全面的反应各种肝纤维化患者的血清标志物。
本发明的另一目的是利用筛选的敏感度、特异度较高的生物标志物制备出检测肝纤维化抗体蛋白芯片。其特点是能够快速高效的显示肝纤维化原因。
本发明还有一目的是利用肝纤维化抗体蛋白芯片的结果建立评分(Scheuer)系统对肝组织活检进行分期。
本发明是通过以下技术方案实现的。
1.建立不同病因肝纤维化患者血清生物标志物的样本库
在全国各省市有代表性的多家医院,患者知情同意,医院伦理审查通过的前提下,收集三年内肝脏穿刺活检并确诊为肝纤维化患者以及100例健康志愿者(肝功正常、肝炎病毒阴性、肝脏影像学无异常)的血液样本,建立样本库。根据临床资料和诊疗经过尽可能将患者分为慢性乙型肝炎(CHB)、慢性丙型肝炎(CHC)、酒精性肝病(ALD)、非酒精性脂肪肝(NAFLD)、肝硬化(LC)。同时将入组患者和健康志愿者的全部医疗信息进行网络登记,统一管理。
2.筛选出敏感度特异度较高的生物标志物
应用RT-PCR和Western-blot分别检测19种已知的有潜能的肝纤维化相关血清生物标志物:前胶原ⅢN-末端肽(PⅢNP)、Ⅰ型前胶原羧基端肽(PICP)、IV型胶原(CⅣ)、层黏连接蛋白(LN)、透明质酸(HA)、糖蛋白YKL-40、MMP-2(白明胶酶A)、MMP-3(基质分解素)、MMP-9(白明胶酶B)、TIMP1(金属蛋白酶组织抑制因子1)、TIMP2(金属蛋白酶组织抑制因子2)、结缔组织生长因子(CTGF)、木糖酰转移酶(XT)、对氧磷酶1(PON-1)、集落刺激因子(CSF)、结缔组织生长因子(CTGF)、血小板衍生生长因子(PDGF)、转化生长因子(TGF-β)、胰岛素样生长因子(IGF-1)在肝纤维化病人及健康志愿者中的基因和蛋白质表达水平,并结合影像学检查结果筛选出敏感度和特异度较高的候选者。应用ELISA技术对所筛选出的的特异标志物进行含量测定。
3.制备对应肝组织活检分期的抗体蛋白芯片
(1)芯片的选择:选用载玻片作为片基,将载玻片硅烷化。
(2)抗体芯片的制备:将上述生物标志物的单克隆抗体通过点阵法在载玻片上进行点样,制备好的芯片放入湿盒中,4℃过夜。
(3)血清样本的标记:将血清样本进行生物素标记,备用。
(4)抗体芯片检测血清样本:将制备好的抗体芯片于室温下封闭1小时,洗涤后将血清样本加样于芯片上,加荧光染料孵育后洗涤,用荧光芯片扫描仪进行信号检测。所得检测数据与ELISA测定的数值相匹配,确定对应的0-4期的荧光信号强度。
4.建立肝纤维化标志物蛋白抗体芯片检测系统
对于进行肝组织活检的肝纤维化患者,根据其Scheuer评分系统的结果0-4期,应用统计学方法找到筛选出来的血清生物学标志物在各个期的差异,从而确定能代表每个期别的最佳血清生物学标志物,并通过大规模的样本库确定合理的数值变异范围。
例如:应用SPSS 25.0统计软件和Med-calc软件进行统计分析。计量资料以均数±标准差表示,采用独立样本t检验;计数资料采用χ2检验。绘制受试者工作特征曲线(ROC曲线)来确定最佳诊断临界值。根据绘制出来的ROC曲线得到筛选出的生物标志物用来区分不同分期肝纤维化患者的临界值,抗体芯片信号值大于该临界值设定为阳性,并得出生物标志物在不同分组间的阳性率,计算不同生物标志物阳性率的差异,从而确定能代表每个期别的最优血清生物学标志物。
本发明的有益的技术效果在于:
本发明建立大规模肝纤维化患者数据库将为纤维化的机制等研究提供资源,同时筛选出敏感度、特异度较高的生物标志物候选者;制备肝纤维化抗体蛋白芯片有助于高通量的检测,降低成本、缩短检测时间;制备的肝纤维化抗体蛋白芯片对应肝组织学活检分期在一定程度上代替肝活检,减少肝活检的需要,降低了取样难度并且减轻了因取样给患者带来的痛苦。
具体实施方式
本发明将通过以下实施例作进一步说明。
实施例1。
制备肝纤维化标志物蛋白抗体芯片检测系统:
(1)芯片的选择:选用载玻片作为片基,将载玻片硅烷化。
(2)抗体芯片的制备:将上述生物标志物的单克隆抗体通过点阵法在载玻片上进行点样,制备好的芯片放入湿盒中,4℃过夜。
抗体蛋白芯片检测血清样本:
(1)血清样本的标记:将血清样本进行生物素标记,备用。
(2)抗体芯片检测血清样本:将制备好的抗体芯片于室温下封闭1小时,洗涤后将血清样本加样于芯片上,加荧光染料孵育后洗涤,用荧光芯片扫描仪进行信号检测。所得检测数据与ELISA测定的数值相匹配,确定对应的0-4期的荧光信号强度。
Claims (1)
1.一种肝纤维化抗体蛋白芯片的制备以及在诊断肝纤维化分期中的应用,其特征在于所述的具体制备及诊断方法如下:
(1)建立不同病因肝纤维化患者血清生物标志物的样本库
在全国各省市有代表性的多家医院,患者知情同意,医院伦理审查通过的前提下,收集三年内肝脏穿刺活检并确诊为肝纤维化患者以及100例健康志愿者(肝功正常、肝炎病毒阴性、肝脏影像学无异常)的血液样本,建立样本库。根据临床资料和诊疗经过尽可能将患者分为慢性乙型肝炎(CHB)、慢性丙型肝炎(CHC)、酒精性肝病(ALD)、非酒精性脂肪肝(NAFLD)、肝硬化(LC)。同时将入组患者和健康志愿者的全部医疗信息进行网络登记,统一管理。
(2)筛选敏感度特异度较高的生物标志物
利用RT-PCR和Western-blot分别检测19种已知的有潜能的肝纤维化相关血清生物标志物:前胶原ⅢN-末端肽(PⅢNP)、Ⅰ型前胶原羧基端肽(PICP)、IV型胶原(CⅣ)、层黏连接蛋白(LN)、透明质酸(HA)、糖蛋白YKL-40、MMP-2(白明胶酶A)、MMP-3(基质分解素)、MMP-9(白明胶酶B)、TIMP1(金属蛋白酶组织抑制因子1)、TIMP2(金属蛋白酶组织抑制因子2)、结缔组织生长因子(CTGF)、木糖酰转移酶(XT)、对氧磷酶1(PON-1)、集落刺激因子(CSF)、结缔组织生长因子(CTGF)、血小板衍生生长因子(PDGF)、转化生长因子(TGF-β)、胰岛素样生长因子(IGF-1)在肝纤维化病人及健康志愿者中的基因和蛋白质表达水平,并结合影像学检查结果筛选出敏感度和特异度较高的候选者。应用ELISA技术对所筛选出的的特异标志物进行含量测定。
(3)制备对应肝组织活检分期的抗体蛋白芯片
A:芯片的选择:选用载玻片作为片基,将载玻片硅烷化。
B:抗体芯片的制备:将上述生物标志物的单克隆抗体通过点阵法在载玻片上进行点样,制备好的芯片放入湿盒中,4℃过夜。
C:血清样本的标记:将血清样本进行生物素标记,备用。
D:抗体芯片检测血清样本:将制备好的抗体芯片于室温下封闭1小时,洗涤后将血清样本加样于芯片上,加荧光染料孵育后洗涤,用荧光芯片扫描仪进行信号检测。所得检测数据与ELISA测定的数值相匹配,确定对应的0-4期的荧光信号强度。
(4)建立肝纤维化标志物蛋白抗体芯片检测系统
对于进行肝组织活检的肝纤维化患者,根据其Scheuer评分系统的结果0-4期,应用统计学方法找到筛选出来的血清生物学标志物在各个期的差异,从而确定能代表每个期别的最佳血清生物学标志物,并通过大规模的样本库确定合理的数值变异范围。
例如:应用SPSS 25.0统计软件和Med-calc软件进行统计分析。计量资料以均数±标准差表示,采用独立样本t检验;计数资料采用χ2检验。绘制受试者工作特征曲线(ROC曲线)来确定最佳诊断临界值。根据绘制出来的ROC曲线得到筛选出的生物标志物用来区分不同分期肝纤维化患者的临界值,抗体芯片信号值大于该临界值设定为阳性,并得出生物标志物在不同分组间的阳性率,计算不同生物标志物阳性率的差异,从而确定能代表每个期别的最优血清生物学标志物。
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