慢性肾衰中医不同证型舌苔中相关蛋白研究

The Study on Associated-proteinum of Tongue Coating of Chronic Renal Failure in Different Syndromes of TCM

目的:研究通过比较慢性肾功能衰竭(CRF,简称慢性肾衰)中医不同证型组患者及正常对照组人群舌苔上清液中蛋白质谱峰的差异,初步探讨慢性肾衰舌苔上清液中相关蛋白变化与慢性肾衰中医不同证型的相关性。方法:临床收集慢性肾衰患者舌苔样本并辨证分型出5个不同证型,其中脾肾气虚组21例、脾肾阳虚组14例、阴阳两虚组18例、脾肾气阴两虚组11例、肝肾阴虚组3例,另有正常对照组38例,对所有样本采用1500r/min的离心转速,离心2min后取上清液,选用NP20蛋白芯片点样,经SELDI-TOF-MS技术测定得到蛋白质谱图,通过Biomarker Wizard软件分析慢性肾衰中医不同证型组及正常对照组之间蛋白质谱图的变化,并利用层次聚类算法进行层次聚类分析和主成分分析(PCA)来比较各组之间蛋白质谱图的差异。结果:(1)应用SELDI-TOF-MS技术筛选出慢性肾衰中医各证型组及正常对照组舌苔样本中有7个差异蛋白质谱峰有统计学意义(P<0.01)。其中,质荷比M/Z1090.66和M/Z1092.68两个差异质谱峰在各证型组均呈高表达;M/Z13302.5一个差异质谱峰在各证型组均呈低表达;M/Z10241.4和M/Z10464.8两个差异质谱峰在脾肾气阴两虚组、肝肾阴虚组高表达,在脾肾气虚组、脾肾阳虚组、阴阳两虚组呈低表达;M/Z3742.33一个差异质谱峰在脾肾气虚组、脾肾气阴两虚组、肝肾阴虚组呈高表达,在脾肾阳虚组、阴阳两虚组呈低表达;M/Z 10867.6一个差异质谱峰在脾肾气阴两虚组呈高表达,在脾肾气虚组、脾肾阳虚组、阴阳两虚组、肝肾阴虚组呈低表达。(2)利用层次聚类算法进行层次聚类分析和主成分分析(PCA),慢性肾衰各证型组之间及与正常对照组舌苔样本之间都有一定的区分度,但均存在部分重叠。结论:该研究从生物学理论的角度初步探讨慢性肾衰舌苔上清液中相关蛋白变化与慢性肾衰中医不同证型的相关性,为中医证候研究提供客观依据。

Objective：Comparing differences of protein spectrum expression in clear supernatant liquid of tongue coating between different syndromes in TCM of CRF patients and normal control group,to explores preliminarily the relationship between change of associated-proteinum in clear supernatant liquid of tongue coating of CRF and different syndromes in TCM of CRF.Methods：Five different syndromes tongue coating samples of CRF patients were collected,among the total samplesthe Qi deficiency of both the spleen and kidney group has 21 samplesthe Yang deficiency of both the spleen and kidney group has 14 samplesthe deficiency of both the Yin and Yang group has 18 samplesthe Qi and Yin deficiency of both the spleen and kidney group has 11 samplesthe Yin-deficiency of both the liver and kidney group has 3 samples,and normal control group has 38 samples.All samples were centrifuged at 1500r/minafter 2mindislodged clear supernatant liquid and spoted one by one to proteinum chips of NP20detected by technique of SELDI-TOF-MS for proteinum spectrograms.Contrasted by software named Biomarker Wizard for the differences of proteinum spectrograms among different syndrome groups and normal control groupand compared differences of protein spectrum expression among different groups by utilizing fuzzy grouping algorithm to run fuzzy grouping analysis and principal component analysis.Results：（1）Tongue coating samples of different syndromes in TCM of CRF and normal control group are determined by technique of SELDI-TOF-MS7 proteinum peaks have statistical significance（P0.05）.The two M/Z1090.66 and M/Z1092.68 show high expression in all syndrome groups;the one M/Z13302.5 show low expression in all syndrome groups;the two M/Z10241.4 and M/Z10464.8 show high expression in the Qi and Yin deficiency of both the spleen and kidney group and Yin-deficiency of both the liver and kidney groupshow low expression in the Qi deficiency of both the spleen and kidney groupdeficiency of both the Yin and Yang group and Yang deficiency of both the spleen and kidney group;The one M/Z3742.33 show high expression in the Qi deficiency of both the spleen and kidney groupQi and Yin deficiency of both the spleen and kidney group and Yin-deficiency of both the liver and kidney groupshow low expression in the Yang deficiency of both the spleen and kidney group and deficiency of both the Yin and Yang group;The one M/Z10867.6 show high expression in the Qi and Yin deficiency of both the spleen and kidney groupshow low expression in the Qi deficiency of both the spleen and kidney groupYang deficiency of both the spleen and kidney groupdeficiency of both the Yin and Yang group and Yin-deficiency of both the liver and kidney group.（2）Using fuzzy grouping algorithm to run fuzzy grouping analysis and principal component analysisamong different syndromes in TCM of CRF and normal control groupthere has discriminationbut partly overlapping.Conclusion：On the point of theory in biologythe study explores preliminarily the relationship between change of associated-proteinum in clear supernatant liquid of tongue coating of CRF and different syndromes in TCM of CRFwhich provides objective evidence for the study of syndrome and penthemeron in TCM.