The Initial Study On Plasma Proteomics Of Kidney-YIN Deficiency

【Background】As one of the basic syndrome,Kidney-yin deficiency is,family history of kidney-yin deficiency medical certification and control of the theory are quite seriously.Especially in recent decades,many scholars use of enzymes,cell biology, immunology and molecular biology,and other modern technologies and methods study found that kidney-yin deficiency syndrome concerned with the hemorheology, endocrine,immune,material and energy metabolism,Trace elements,biochemistry, enzymes and abnormal gene expression,and so on,confirmed its preliminary modern basis of the pathophysiology,and some of its interpretation of certain details of the theory of Chinese medicine and science,have made certain achievements.cDNA Lib of Kidney-yin deficiency(including diabetes,chronic nephritis, lupus nephropathia and sub-health)was successfully set up in our prior study works by some techniques and methods,such as RNA microamount-amplification, differential hybridization and gene cloning.We also found that fourty six genes were closely related with Kidney-yin deficiency in our prior study about gene array based on the theory of Chinese medicine measuring Chinese syndrome.It is well known that a gene should express a certain protein to take a part in some biological action.Type and degree of gene expression are affected by both outside environment and internal condition.The study of proteome is exactly aimed to found internal changes of organism in morbid condition.The changes is focused on the specificnesses and differences of protein expression.On the base of the specificnesses and differences resulted from making use of techniques of proteome, a general regularity of syndrome can be summed up to analyze Kidney-yin deficiency in all aspects.The blood plasma is a very good sample which is charactered with obtaining easily,being used widely and protein containing highly.The expression level, post-translational modification and post-translational interaction of protein in blood plasma can be studied in the whole by the techniques of proteome.Then the disease process and medicine therapy will be well known from results of the studies which is very important in foundation study and exploratory development.【Object】The study is aimed to find different proteins between patients of Kidney-yin deficiency and healthy people and approach the machenism of Kidney-yin deficiency in protein level.【Methods】1.The samples were normal human plasmas.The samples were treated with three different ways:removing albumin and IgG globulin,boiling and no treating. Then all samples were analyzed by two-dimensional gel electrophoresis in different pH grad.After Staining by argenti nitras,the gels were scaned and analyzed by PDQuest software.According to the results,the best method of two-dimensional gel electrophoresis was established. 2.The patients with Kidney-yin deficiency were divided into experimental group.While the patients with Kidney-yang deficiency were divided into negative control group and those healthy people were divided into normal control group.All the plasma samples in different treating ways were analyzed by two-dimensional gel electrophoresis.3.Eighteen clear points were chosen from the gels to be identified and analyzed by the method of PMF in mass spectrogram.The points being picked up should be clear and specific.【Results】1.The electrophoregram of normal human plasmas was obtained by two-dimensional gel electrophoresis.By silver nitrate staining,the proteins SDS hot plasma sample of the entire two-dimensional gel electrophoresis staining Deep, scattered clear,and to avoid in the albumin/IgG globulin in the process of removing the low abundance of non-specific absorption caused by man-made differences.The electrophoregram with high-resolution and good-reproducibility was the foundation for choosing good protein points.2.With two-dimensional electrophoresis technology to sub-health state,IgA nephropathy and chronic kidney-yin deficiency card glomerulonephritis,a deficiency syndrome and normal expression of the difference between two-dimensional gel electrophoresis and plasma digital image analysis,and the normal Compared to people:①There are 73 spots which expression levels were increased in the plasma protein of the sub-health kidney-yin deficiency,and 90 spots were decreased.10 protein spots specific appeared,14 protein spots missed.About kidney-yang deficiency of sub-health,the expression levels of 67 protein spots were increased, and 191 protein spots were decreased.The number of specific protein spots was 17, the missing protein spots was 19.②There are 109 spots which expression levels were increased in the plasma protein of the Chronic Glomerulonephritis kidney-yin deficiency,and 82 spots were decreased.36 protein spots specific appeared,9 protein spots missed.About kidney-yang deficiency of Chronic Glomerulonephritis, the expression levels of 112 protein spots were increased,and 79 protein spots were decreased.The number of specific protein spots was 39,the missing protein spots was 14.③There are 122 spots which expression levels were increased in the plasma protein of the IgA nephropathy kidney-yin deficiency,and 120 spots were decreased.21 protein spots specific appeared,19 protein spots missed.About kidney-yang deficiency of IgA nephropathy,the expression levels of 131 protein spots were increased,and 111 protein spots were decreased.The number of specific protein spots was 18,the missing protein spots was 221.3.The above results,twenty-nine protein points in Kidney-yin deficiency group were different from control groups.Among all these different points,nine protein points showed high-expression and eleven showed low-expression.Seven protein points were only found in healthy people and six protein points were only found in patients with Kidney-yin deficiency.4.Seven proteins were found to be closely related with Kidney-yin deficiency through the techniqueof two dimensional gel electrophoresis and MALDI-TOP mass spectra.Among these seven proteins,four proteins,includingα1-microglobulin, retinol binding protein,tetraiodothyronine transferring protein and heat shock protein,were highly expressed in Kidney-yin deficiency group.The other three proteins,including H-chain of fibrinogen,α1-antitrypsin and complement C4-B, were highly expressed in normal control group.The function of the different proteins was related with hormonal modulation,immune response,oxidative stress,signal conduction and cystoskeleton construction. 【Conclusions】1.Because of little-losing,simple and good repeatability,the method of two-dimensional gel electrophoresis using heating-SDS whole plasma sample is an effective means of the syndrome plasma proteomics research.2.In the plasma proteomics research,we screenned and identificated seven plasma specific differences proteins of the kidney-yin deficiency card of a kidney-yin.It was the foundation on to explore the kidney-yin deficiency mechanism in the protein level.