| ObjectiveTraditional Chinese medicine(TCM)constitution research has developed for more than two thousand years and is an important part of TCM.The influence and contribution of TCM constitution theory clinical practice in the direction of health care,preventive and rehabilitation medicine have become increasingly prominent,as well as a key discipline integrating TCM with mordern medicine,and striving to promote the development of the large health industry.In contrast to clinical popularization,the relatively weak basic research has greatly limited the integration process of TCM physiology and modern medicine.With the rapid development of omics technology,people have a clearer and deeper understanding of life phenomena.As an executor of life activities,proteins have played an important role in the occurrence and development of diseases,proteomics has become a powerful tool for life science research.Therefore,to explore and analyze the biological basis for the constitution theory of TCM and parsing the objective reality of clear constitution phenomenon of traditional Chinese medicine,enriches the modern scientific connotation of TCM constitution theory,guides clinical practice in multiple directions,levels and points,promote the further development of TCM Constitution,and improve the quality of life of the masses.In this study,two commonly used techniques in proteomics--gel electrophoresis combined with mass spectrometry and antibody array technology,were used to identify and compare serum protein profile of different constitutiona populations,in order to discover the expression characteristics of TCM constitutional phenomena at the protein level and explain the cause and formation of constitutional phenomena,to judge the change trend of the constitution type,and to construct a digital model to classify the constitution according to differentially expressed proteins,to provide experimental evidence and potential reference indicators for basic research and clinical diagnosis and treatment of TCM constitution.MethodsThis project is divided into three parts.The first part is a statistical analysis of the characteristics of a total of 947 people in traditional villages in Lingnan area from 2015 to 2016.Mainly to analyze the basic information of the traditional village population,including the classification of TCM constitutions,physiological indicators,and quality of life assessment.Statistical description and comparison of differences among TCM constitution groups were shown.The second part uses blue native polyacrylamide gel electrophoresis(BN PAGE)combined with ultra performance liquid chromatography-electrospray ionization-mass spectrometry/Mass spectrometry(UPLC-ESI-MS/MS)to study the serum protein expression difference among people with different TCM constitution in Lingnan area.To study the characteristics of protein expression profiles in 139 serum samples from 19 healthy individuals and 120 individuals with dyslipidemia that 30 induvidules in each group of Balaced constitution,deficient kond constitution,excessed kind constitution and constitution combined deficiency with excess.BN PAGE was used to separate serum protein complexes of different constitution populations,and imageJ software was used to identify complex gel bands,and clustering and clinical information correlation analysis were performed based on the band characteristics.Protein complex bands were identified by mass spectrometry to find the potential specific proteins or protein complexes of different constitutions.We also study individual differences from the complex band type of Balanced constitution population.In the third part,the high-throughput protein antibody anssay technology was used to compare the serum protein expression difference between people with Balanced constitution and who with“Dampness”kind constitutions.Detecting serum samples from Balanced,Phlegm-dampness and Dampness-heat populations,to find differentially expressed proteins and related biological processes of three constitutional populations.Highthroughput protein chips to detect serum protein profile of Balanced constitution and "Dampness" kind constitutions,and transformation constitution,looking for potential the biomakers before and after the outcomes of the two constitutional populations.Screen out sevaral differential proteins to build a support vector machine(SVM)data model for serums of Balanced constitution and "Dampness" kind constitutionsResults(Ⅰ)Overall characteristics of the village population in Lingnan.1.A total of 947 health records were established,with 320 men and 628 women.The minimum age is 18 years,the maximum age is 91 years,the average is 54 years,and the median is 57 years.2.TCM Constitution Assessment:A total of 921 people finished TCM constitution assessment(Table 1).#123.Blood detected disease921 people participated in the determination of TCM constitution type.A total of 842 people completed blood tests,of which 686(81.47%)people had dyslipidemia,306(36.34%)people had hyperuricemia,122(14.49%)had renal insufficiency,6 people had hyperfunction(0.71%),74 people had diabetes(8.79%),and 31 had anemia(3.68%).4.Dyslipidemia index(?)#12(Ⅱ)Characteristic expression profiles of serum protein complexes in people of different constitutions1.The gel band pattern of protein complex is stable,and the population can be classified according to the pattern.The protein complexes of 139 serum samples were separated by BN PAGE,which contained low and middle abundance proteins,they were presented in the form of gel bands.BN PAGE marker was used as a reference to cluster the samples into 2-5 gel patterns.In general,the gel patterns of serum samples from different constitutions were similar.But it is also possible to distinguish between Balanced and unbalanced constitution populations according to the gel band type.2.There are specific proteins related to lipids in protein complexes.The protein in the protein complex band was identified by mass spectrometry,which confirmed that BN PAGE technology had high repeatability and stability,and could separate different serum protein complexes.The serum of dyslipidemia patients is enriched to lipid related protein complexes,including 8 proteins such as ApoE,PON1 and other lipid transport,metabolism and other related proteins.The protein complex from the gel bands was recovered,and western blot experiments on lipid-related proteins was performed.It was found that the expression level of ApoE in some bands was related to constitution type and age,and the expression level or proportion of PON1 in some bands was related to high density lipoprotein cholesterol(HDLC)or low density lipoprotein cholesterol(LDLC).3.The internal differences of the people with Balanced constitution maybe related to the tendency of constitution transformation.The gel patterns of two major types(type A and type B)were identifiedand analyzed by mass spectrometry.The results of mass spectrometry indicated that each protein complex consisted of 48-87 different proteins.The different complex bands had similarity and specificity.The A type specific protein was related to the biological process of lipid metabolism,and the B type specific protein was related to complement activation.Type A sample population is relatively high in Qi-deficiency conversion score,and type B sample population has higher Dampness-heat score,the difference is statistically significant.(Ⅲ)Differential protein expression profiles in the Balanced and"Dampness" kind populations1.Comparing the differential proteins of Balanced,Phlegm-dampness and Dampness-heat constitution,among which differentially expressed proteins were related to metabolic diseases and cardiovascular and cerebrovascular diseases.A total of 178 differential proteins were screened from 3 pairs of Dampness-to-Balanced constitution serum samples,and a total of 79 differential proteins were screened from 4 pairs of Balanced-to-Dampness constitution serum samples.There were 19 differential proteins in the Balanced and "Dampness" kind population.2.Construction of SVM classification model of "Dampness" constitution with high accuracy,specificity and sensitivity.Seven different proteins TACI,Siglec-7,Dectin-2,AIF,TROP-2,MMP-2 and Legumin were selected to construct the SVM model and verify the Balanced and Dampness constitution population.The model can effectively classified of constitution population.3.Eating and sleeping habits have a greater impact on people with Dampness constitutions.Construction of a differential co-expression network for Balanced and Dampness constitutions.The differential protein expression levels in Balanced and Dampness constitutions have significant correlation with clinical information,mainly related to dietary habits and other lifestyle habits.Differentially expressed proteins are mainly involved in biological functions related to the body’s inflammatory response and immune regulation.Conclusion(Ⅰ)The kind of Dampness constitution is the main type of unbalanced constitutions in the population of Lingnan area.By serum proteomics technology,we can accurately study the potential specific proteins of different population of traditional Chinese medicine in Lingnan area and predict their related biological pathways.The dual research methods of mass spectrometry and chip technology can complement each other and take into account the breadth and depth of proteomics research.To realize the exploration and specific research on the material base of traditional Chinese medicine.(Ⅱ)The traditional Chinese medicine constitutional structure characteristics of the population in the traditional villages of Lingnan area are:Balanced constitution are the main type,and most of the people in the constitution have more than two constitutions.High detection rate of metabolic diseases exist.(Ⅲ)Research system based on BN PAGE technology for the protein complex in sera of people with dyslipidemia was established,which effectively enriches low-abundance proteins with biological functions in blood and presents them as protein complexes.It is the first explicitly proposed that specificities exist in blood functional protein complex,and proved its correlation with dyslipidemia.Blood protein comple x expression profile of people with different dyslipidemia in traditional Chinese medicine are relatively stable and repeatable.Different constitutional people can be classified.It established a link between the traditional Chinese medicine theory emphasizing function and phenotype and the proteomics research based on molecular biology.(Ⅳ)The main differential protein functions in the serum of Balanced and"Dampness" populations are concentrated in the inflammatory response and immune function.This may be the biological basis of the differences between the Balanced and Dampness people.The formation of Dampness constitution may be directly related to diet,siesta habit,physical activity and other lifestyle.The relationship between the function of differential proteins and cardiovascular and cerebrovascular diseases and metabolic diseases is explained,which further confirmed the relationship between TCM constitution and disease susceptibility. |
PDF Full Text Request