| On the bases of the establishment of rat model with Qi-stagnancy/Qi-deficiency and blood stasis,miniature swine model with Syndrome of Phlegm and Stagnated Blood,the present research employed the two dimensional gel electrophoresis(2-DE) and the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) to explore the proteomic profiles in the syndrome of Qi-stagnancy and blood stasis,Qi-deficiency and blood stasis and syndrome of phlegm and blood stasis of coronary heart disease.The protein in serum,blood vessels and myocardial tissue from the normal and three different models was respectively extracted,isolated,and identified to in the research.By a comparative proteomics approach we compared protein expressing pattern in normal and syndrome animals.The confirmed specific protein expressions associated with the syndrome of blood stasis will be recognized as the potential diagnostic biomarker and the therapeutic target,and will be helpful to uncover the molecular mechanisms of the processes in the syndrome of blood stasis.Part One Primary Serum Proteomies Analysis of Rat Model with Syndrome of Qi-stagnancy and Blood StasisThe rats were randomly divided into normal and model group with Qi-stagnancy and blood stasis,10 in each group.The rat serum from the same group was mixed and albumin was removed.IPG stripe(24cm,pH4-7) was used in isoelectric focusing electrophoresis and each experiment was repeated 3 times for better repeatability.Serum protein expressing pattern in normal and model group was compared by Image Master 2D Elite 6.01 analysis software.The results showed that under the same conditions,347±37 and 354±20 proteins had been observed in model group and the normal group.After one gel was chosen as the reference,the other gels were matched with the reference one.The average NbMatches was 280±23,the PercentMatches was 75.3%.Among the protein spots showing 2 or more than 2 fold difference in protein volume percentage(%Vol),compared with normal group,7 changed proteins had been determined:1 protein down-regulated,5 proteins up-regulated and 1 protein lost in rat serum of model group.These differential proteins spots were excised from preparative gels and digested into peptides.These peptides were analyzed by MALDI-TOF-MS.Data were then searched with the search engine MASCOT against a NCBI nonredundant protein sequence database.The cited protein scores greater than 71 were credible.2 proteins were identified by MALDI-TOF-MS,in which Isovaleryl-Coa Dehydrogenase was down-regulated,and STAT3-interacting protein was up-regulated.To our best knowledge,relevant reports with these two proteins are rare,STAT3-interacting protein is closely related to cytokine signaling pathway.How they play the role in the formation of the syndrome of Qi-stagnancy and blood stasis needs further study.Part Two Primary Serum Proteomics Analysis of Rat Model with Syndrome of Qi-deficiency and Blood StasisThere are 10 rats in the normal group and the rat model group with Qi-deficiency and blood stasis.We compared serum protein expressing pattem in nomal and model group by Image Master 2D Elite 6.01 analysis software.The results showed that 413±13 and 401±37 proteins had been observed in serum of model and normal group.Matched with the reference gel,the average NbMatches was 368±12,the PercentMatches was 88.5%.Among the protein spots showing 2 or more than 2 fold diffenerce in%Vol of protein,compared with normal group,7 changed proteins had been determined:1 protein down-regulated,5 proteins up-regulated and 1 protein lost in rat serum of model group.By MALDI-TOF-MS and then with the search engine MASCOT,6 proteins were identified,in which CD5 antigen-like was down-regulated,other 5 proteins including haptoglobin,complement component C3,etc.were up-regulated.The functions and features in these proteins had relevance to the pathological process of inflammation,immune regulation,which is consistent with the known occurrence and development of the syndrome of Qi-deficiency and blood stasis.Part Three Proteomics Research of Experimental Mini-pig Model with Syndrome of Phlegm and Blood Stasis of Coronary Heart Disease12 mini-pigs were used in the research,6 for normal group and the other 6 for model group with phlegm and blood stasis of coronary heart disease.The model was established in mini-pig by high fat diet combined with balloon injury.Serum,coronary artery and myocardial tissues were all separated by two dimensional gel electrophoresis,using IPG stripe(24cm,pH4-7) and 12.5%SDS-PAGE,repeatedly 3 times.We compared protein expressing pattern in normal and model group by Image Master 2D Elite 6.01 analysis software.1.In serum protein profile,average 3944±23,440±25 proteins had been respectively observed in the model group and the normal group.Matched with the reference gel,the average NbMatches was 363±25,the PercentMatehes was 82.8%;compared with normal group,17 changed proteins had been determined:1 protein down-regulated,6 proteins up-regulated,7 proteins lost and 3 proteins specific expressed in mini-pig serum of the syndrome of phlegm and blood stasis of coronary heart disease.2.In 2-DE coronary artery tissue protein profile,average 1098±37,967±44 proteins had been observed in the model group and the normal group.Matched with the reference gel,the average NbMatches was 895±45,the PercentMatches was 86%;compared with normal group, 35 changed proteins had been determined:14 proteins up-regulated,17 proteins specific expressed and 4 proteins were modified in mini-pig coronary artery tissue of the syndrome.3.In 2-DE myocardial tissue protein profile,average 978±32,890±38 proteins had been observed in the model group and the normal group.The average NbMatches was 725±43,the PercentMatches was 76.7%;compared with normal group,14 changed proteins had been determined:13 proteins up-regulated and 1 proteins specific expressed in mini-pig myocardial tissure of the syndrome.By MALDI-TOF-MS and then with the search engine MASCOT,3 proteins in serum sample were identified,including complement component 4 binding protein,apolipoprotein E precursor etc.And 18 proteins in coronary artery tissue were identified,that was transgelin,coactosin-likel etc.As for the myocardial tissue,9 proteins including HSPA8 protein,apolipoprotein A-Ⅳetc,were identified.More than half of the above identified proteins were classified as albumin,which involved in the pathological process of inflammation,vascular remodeling and lipid metabolism etc.These proteins may play an important role in process of the formation,occurrence and development of the syndrome of phlegm and blood stasis of coronary heart disease.Conclusion1.The protein profile of syndrome of Qi-stagnancy and blood stasis,Qi-deficiency and blood stasis rat serum displayed obviously difference compared with normal rat serum.The results implied that various different proteins led to the development of these two syndromes.2 and 6 different proteins were identified in rat serum of syndrome of Qi-stagnancy and blood stasis, Qi-deficiency and blood stasis,which might be related to the inflammation and immune modulations during the development of these two syndromes.2.Compared with normal healthy mini-pig,the protein profile of the model mini-pig serum, coronary artery and myocardial tissue all demonstrated obviously difference.Total 30 different proteins were identified in three different samples,which could participate in the processes of inflammation,vascular remodeling,and lipid metabolisms in the syndrome of phlegm and blood stasis of coronary heart disease.3.The identified proteins in the above syndrome models,for one thing,constitute the evidence that these animal model have coincidence with clinical features of the disease,and for another thing,indicate that part of different proteins definitely have close relation to the formation of these three syndromes of blood stasis.Certain proteins maybe become potential and valuable clinically molecular markers can be used for clinical diagnosis and prediction of these three syndromes of blood stasis.
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