肝硬化正虚血瘀证候与血清蛋白质组学所见

作者	单位
周烨威	上海中医药大学附属曙光医院
徐品初	肝肾疾病病证教育部重点实验室(上海中医药大学) 上海中医药大学肝病研究所
徐列明	上海市中医临床重点实验室国家中医药管理局重点研究室(慢性肝病虚损)
成扬	上海市中医临床重点实验室国家中医药管理局重点研究室(慢性肝病虚损)

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中文摘要:

目的从肝硬化患者证候和血清蛋白质组学两方面,探讨肝硬化中医正虚血瘀的病机。方法采集到肝硬化男性患者44例和健康男性志愿者17名的四诊信息和血清标本。对肝硬化患者的四诊信息进行辨证和证候频率分析,归纳为不同证候群。运用Cu螯合磁珠与基质辅助激光解析电离飞行时间质谱技术检测患者和健康者血清蛋白质组,运用Bruker公司统计学分析软件分析,找出不同证候患者之间及与健康对照组之间有显著差异的蛋白质峰,运用QC算法建立肝硬化不同证候的分类预测模型。结果肝硬化证候出现频率超过30%、由高到低依次排列的是:乏力、精神萎靡、蜘蛛痣、肝掌、纳差、齿龈鼻肌衄、脘腹胀满、动则气促、面晦黯、胁肋刺痛、腰膝痠软、胁肋隐痛、五心烦热或低热、潮热盗汗。脾气虚弱证候组17例,其中属Child-Pugh A级的例数占64.7%;肝肾阴虚证候组12例,其中属Child-Pugh C级的例数占66.7%;血瘀证候组15例,在Child-Pugh A级和C级中所占比例相似,均>40%。在Child-Pugh A级出现的蜘蛛痣、肝掌、动则气促、五心烦热或低热、潮热盗汗、腹壁脉络曲张、下肢水肿等证候,在Child-Pugh C级出现的频率增加,差异有统计学意义(均P<0.05)。选择峰值明显质荷比为4642.81、4963.91、5247.8、5805.95、6305.27和12447.7的蛋白质峰构成脾气虚弱证候诊断模型中的特征蛋白质峰,前5个峰在Child-Pugh A级和C级的蛋白质峰谱中都能找到;9290.3蛋白质峰为肝肾阴虚证候诊断模型的特征蛋白质峰,该峰与7768.29蛋白质峰是肝肾阴虚证候组与其他2证候组比较均下调的蛋白质峰,均包括在乙肝肝硬化的诊断模型中,但未出现在Child-PughA级的蛋白质峰谱中,而在Child-Pugh B级和C级的蛋白质峰谱中表达逐级下调。另外17个在肝肾阴虚证候患者表达有明显变化的蛋白质峰中,8个在Child-PughA级中均有表达;在Child-Pugh A级和C级中均表达的4964.55和5806.83蛋白质峰构成乙肝肝硬化血瘀证候诊断模型中的特征性蛋白质峰。建立的乙肝肝硬化脾气虚弱证候诊断模型,敏感度为100%,特异度为82.35%;建立的肝肾阴虚证候诊断模型,敏感度为100%,特异度为94.12%;建立的血瘀证候诊断模型,敏感度为100%,特异度为100%。结论正虚血瘀是肝硬化全程均有的基本病机,"正虚"包括了脾气虚弱和肝肾阴虚。肝肾阴虚虽在肝硬化失代偿期明显,但在代偿期已有表现,需尽早顾护。

英文摘要:

Objective To study the basic pathogenesis of "asthenia of healthy energy and blood stasis" in liver cirrhosis studied by Chinese syndromes and serum proteomics. Methods The information of four methods of examinations and serum samples were collected from 44 cases of male cirrhotic patients and 17 cases of healthy male volunteers. The different syndrome groups were summarized according to syndrome differentiation and frequency analysis using the patient′s information of four methods of examinations. The serum proteins were isolated by magnetic beads and detected by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The proteins expressed differently between cirrhotic patients of different syndrome types and healthy volunteers were analyzed by statistical analysis software (product of Bruker Corporation ClinProTools 2.1 software). The diagnosis model was established by QC algorithm. Results The liver cirrhosis syndrome with the appearance frequency of more than 30% was sequenced from high to low as fatigue,listlessness,spider telangiectasia,liver palms,anorexia,bleeding from the nose,the gum or the subcutaneous tissue,the abdominal distention,shortness of breath while moving,dim facial complexion,pricking pain of the flank,weak waist and knees,dull pain in the flank,burning sensation of five centers,or low fever,hectic fever,and night sweat. The cases belonging to Child-Pugh A in the seventeen patients of the Pi-qi asthenia syndrome group accounted for 64.7%. The cases belonging to Child-Pugh C in the twelve patients of the Gan-Shen yin deficiency syndrome group accounted for 66.7%. The cases belonging to Child-Pugh A were similar to the cases belonging to Child-Pugh C in the fifteen patients of the blood stasis syndrome group,being more than 40%. Such syndromes as spider telangiectasia,liver palms,shortness of breath while moving,burning sensation of five centers,or low fever,hectic fever,and night sweat,varicose vein of the abdominal wall,and edema of lower extremities appeared more frequently in Child-Pugh C than in Child-Pugh A (all P<0.05). The characteristic protein expression peak with mass-to-charge ratio of 4 642.81,4 963.91,5 247.8,5 805.95,6 305.27,and 12 447.7 in the Pi-qi asthenia syndrome diagnosis model were chosen. The former five peaks could be found in Child-Pugh A and Child-Pugh C. The protein expression peak with mass-to-charge ratio of 9 290.3 was the characteristic protein expression peak in the Gan-Shen yin deficiency syndrome diagnosis model. The protein expression peak with mass-to-charge ratio of 9 290.06 and 7 768.29 were down-regulated in the Gan-Shen yin deficiency syndrome group compared with the other two syndromes groups. The protein expression peaks 9 290.3 and 7 768.29 were included in the diagnosis model of hepatitis B cirrhosis. They did not appear in Child-Pugh A,while they were gradually down-regulated in Child-Pugh B and Child-Pugh C. Of the other seventeen protein expression peaks in patients of the Gan-Shen yin deficiency syndrome,eight expressed in Child-Pugh A. The protein expression peaks 4 964.55 and 5 806.83 that expressed both in Child-Pugh A and Child-Pugh C constituted the characteristic protein peaks of the hepatitis B cirrhosis blood stasis diagnosis model. The diagnosis model of the Pi-qi asthenia syndrome was established with the sensitivity of 100% and the specificity of 82.35%. The diagnosis model of the Gan-Shen yin deficiency syndrome was established with the sensitivity of 100% and the specificity of 94.12%. The diagnosis model of the blood stasis syndrome was established with the sensitivity of 100% and the specificity of 100%. Conclusions Asthenia of healthy energy and blood stasis was the basic pathogenesis during the whole process of liver cirrhosis. Asthenia of healthy energy covers Pi-qi asthenia and Gan-Shen yin deficiency. Gan-Shen yin deficiency was obvious in the compensation stage of liver cirrhosis,but it has manifested in this stage. So early treatment was necessary.

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