The Study On The Theory Of "Kidney Governing Bone" Based On Differential Proteome Of Bone Tissue In Patients With Kidney-Yin Deficiency,Kidney-Yang Deficiency

ObjectiveTo compare the differences of proteome map by protein two-dimensional gel electrophoresis(2-DE)technology from cortical bone and cancellous bone proteomics of kidney-yin deficiency,kidney-yang deficiency and non-kidney deficiency group,and to clarify the protein related to the occurrence of kidney-yin deficiency and kidney-yang deficiency syndrome.Under using the Western Blot and RT-PCR to examine the identification of some differential proteins,and serum ELISA was used to screen the differential proteins that were consistent with the expression of bone tissue to elucidate the scientific connotation the theory of "Kidney governing bone" from the level of bone tissue protein in patients and to provide experimental basis of the potential serum markers and rich diagnostic criteria for kidney-ying deficiency and kidney-yang deficiency.Method1.Human cortical bone and cancellous bone were obtained from kidney-yin deficiency,kidney-yang deficiency and non-kidney deficiency group.The total protein was extracted and prepared.Two-dimensional gel electrophoresis(2-DE)technique was used to obtain human cortical bone and cancellous bone Bone proteomics map.2.Using the PD Quest software to analyze the histological analysis of the human bone proteomics of kidney-yin deficiency,kidney-yang deficiency and non-kidney deficiency group,and to control the differentially expressed protein spots with the protein volume and expressed protein spots were targeted by change of 1.5 times as the standard.3.The proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS)and the bioinformatics database was retrieved to identify and analyze the difference protein name and its function.4.In the preliminary basis for determining differences in protein function,combined with the model of bone tissue tutor team early kidney yin deficiency and kidney yang deficiency rats proteome research results,research significance between group differences in common protein on the theory of "kidney dominating bone",and later serum markers screening according to the factors such as kidney yin deficiency,so selected kidney yang deficiency group a total of 6 differentially expressed proteins.Western Blot and RT-PCR were used to detect the expression of a-enolase(Enol),mitochondrial heat shock protein 70(Hsp70),apolipoprotein AI(ApoA-?),cancellous bone osteoprotegerin(OPG),bone morphogenetic protein 4(BMP-4)and GDP dissociation inhibitory factor ?(Arhgdib)were verified.5.ELISA was used to detect the expression of the related proteins in the respective serum samples.6.All data was used by SPSS 19.0 software for statistical analysis.Statistical data are expressed as mean ± standard deviation(x ±S).Compared with the one-way ANOVA and LSD test,there was significant difference between the two groups(P<0.01).There was significant difeference between the two groups(P<0.01).Result1.The protein profile of the bone tissue samples of kidney-yin deficiency,kidney-yang deficiency and non-kidney deficiency group was clear,the protein spots were well identified,the consistency of the group was uniform,and the human cortical bone and cancellous bone were different The number of protein spots was 490 and 582 respectively.2.The protein profiles of the bone tissue of the patients with kidney-yin deficiency,kidney-yang deficiency and non-kidney deficiency group were compared by mass spectrometry.Bioinformatics showed that the protein was:Kidney-yin deficiency syndrome with the following 26 closely related proteins:? 7 expression of protein exists in cortical bone and cancellous bone:alpha enolase,apolipoprotein A-I,osteopontin and bone morphogenetic protein 2 and 4 proteins were up-regulated,GDP dissociation inhibitor beta and translation control tumor protein and bone morphogenetic protein 4 and 3 proteins were downregulated;?Vimentin and collagen type I alpha chain-1 deoxyuridine three phosphatase,vitamin D binding protein 3 and 4 up-regulated the expression of cortical bone in mitochondrial protein,heat shock protein 70,proliferating cell nuclear antigen,heart muscle nutrients like cells and mitochondrial factor 1 heat shock protein 90 and 4 downregulated proteins;? The cancellous bone in annexin A1,beta tubulin 2C,mitochondrial heat shock protein 60,pyruvate kinase,osteoclastogenesis inhibitory factor and insulin Insulin-like growth factor binding protein 3 expression 6 up-regulated protein,calreticulin 3,heat shock protein B2,N in the middle end of osteocalcin,peroxisome proliferator activated receptor coactivator-1,steroid receptor coactivator 3 5 downregulated proteins.The occurrence of Kidney-yang deficiency syndrome is closely related to the following 24 differential proteins:? Co-existence of seven identical proteins:mitochondrial heat shock protein 90 up-regulation,bone morphogenetic protein 4,osteopontin,GDP dissociation inhibitory factor(3,tyrosine kinase receptor,pyruvate kinase isoenzyme,protein disulfide isomerase A3 and other 6 protein expression down;? Cortical bone apolipoprotein AI,alpha enolase,mitochondrial heat shock protein 70 et al 3 proteins were up-regulated,annexin Al,annexin A3,peroxisome proliferator-activated receptor gamma coactivator-1,type I collagen cross-linked amino-terminal peptide and bone morphogenetic protein 2;? The expression of anti-tartrate acid phosphatase,mitochondrial heat shock protein 60,osteoclastogenesis inhibitory factor and vimentin protein in cancellous bone were up-regulated,1 chain,mitochondrial ATP synthase subunit a,calcein 3,myogenic myosin a-4 and so on.There were 23 differentially expressed proteins in the bone tissue of kidney-yin deficiency and kidney-yang deficiency group.The main contents were as follows:? cortical bone,kidney-yin deficiency group,mitochondrial heat shock protein 70,mitochondrial heat shock Protein 90,mitochondrial ATP synthase subunit a,ankyrin repeat,small molecule heat shock protein and bone morphogenetic protein 4 were up-regulated,osteopontin,a-enolase,peroxidative protein 2,Fibroblast growth factor receptor 3,osteosalic acid protein,GDP dissociation inhibitory factor(3 and other 6 protein expression;? cancellous bone,kidney-yin deficiency group as a reference,kidney-yang deficiency group of tartrate acid phosphatase,Mitochondrial heat shock protein 60,ankyrin repeat sequence and transforming growth factor-a were up-regulated,osteosalic acid protein,pyruvate transdermase isoenzyme,annexin A3,peroxidative protein 2,protease The expression of 7 proteins,?-subunit 9,phosphoglycerate kinase 1 and bone morphogenetic protein 4 were down-regulated.3.Exclude the above 20 cortical bone and cancellous bone in the repeated expression of differentially expressed bone protein,combined with bioinformatics search results,there are 41 different proteins left.According to the bioinformatics search,most of these proteins are closely related to the function of bone tissue,bone metabolism and transport of organic matter in bone tissue,such as type ? collagen ?-1 chain,bone morphogenetic protein 4 Protein and fibroblast growth factor receptor 3 and other proteins can play a catalytic role in the process of bone cell metabolism,regulation and other effects;vitamin D binding protein 3,calcein 3 and other proteins are closely related to material transport."Kidney" may play a "governing bone" role by regulating these proteins that are closely related to bone tissue.4.Western Blot and RT-PCR showed:compared with non-Kidney-deficiency group,the expression of Enol and ApoA-I in cortical bone of Kidney-yin deficiency group was higher than that of cortical bone,and cortical bone Hsp70,cancellous bone BMP-4,Arhgdib and OPG were low expression.ApoA-I was highly expressed,cancellous bone BMP-4,Arhgdib,OPG was low expression.Compared with the kidney-yin deficiency group,the expression of Hsp70 and cancellous bone in the cortical bone of the kidney-yang deficiency group was higher than that in the cortical bone,and the apoptotic BMP-4 was higher in the cortical bone,while ApoA-I and cancellous bone Arhgdib There was no significant difference.5.The results of ELISA showed that the levels of ApoA-I and OPG in the Kidney-yin deficiency group were significantly lower than those in the non-renal deficiency group,and the contents of ApoA-I,BMP-4 and Arhgdib Content decreased,Enol,OPG and Hsp70 content increased.Compared with kidney deficiency group,Hsp70 increased in kidney-yang deficiency group,BMP-4 and Arhgdib decreased.The expression of Hsp70 and BMP-4 in serum was consistent with that of bone.The serum ApoA-I in kidney-yin deficiency group was consistent with that in bone tissue,while the serum and bone tissue of kidney-yang deficiency group were different.The results of Enol,Arhgdib and OPG in virtual group were inconsistent with their expression in bone tissue.Conclusion1.Compared with non-kidney patients with bone tissue,kidney-yin deficiency cortical bone type I collagen a-1 chain,deoxyuridine triphosphatase and other 15 different proteins,cancellous bone Annexin Al,? tubulin 2C,etc.18 differentially expressed proteins,cortical bone morphogenetic protein 4,osteopontin and other 15 differential proteins,cancellous bone mitochondrial heat shock protein 60,osteoclastogenesis inhibitory factor and other 16 differentially expressed proteins,Most of these are related to bone structure,bone metabolism regulation and material transport and other functions,suggesting that these proteins may be involved in the "kidney bone" process,"kidney" on the expression of these proteins different regulation,may be "kidney bone "One of the scientific connotations of theory.2.The difference of mitochondrial heat shock protein 90,mitochondrial ATP synthase subunit a and so on in cortical bone showed that there were differences in expression of bone protein in osteoblasts and osteoblasts Generation of inhibitory factors such as 11 there are differences in expression,these are kidney-yin deficiency,kidney deficiency syndrome is one of the micro-essence,kidney-yin deficiency,kidney-yang state bone structure and functional differences in the material basis.3.The expression of Hsp70 was low and the expression of ApoA-I was high in kidney-yang deficiency state.The expression of Hsp70 was high in the kidney-yang deficiency state,All the expression of protein was confirmed by 2-DE,Western Blot,RT-PCR and serum ELISA,and they could be used as the potential marker of kidney-yin deficiency and kidney-yang deficiency's serum.