| Objective: To investigate the effect of the factors on the difference of the expression of the protein to the spleen in the normal rats and the spleen in the spleen yang deficiency rats. To explore the mechanism of pathogenic dampness.Methods:1.The 40 SD rats were randomly divided into 4 groups: control group, spleen yang deficiency group,external damp group and spleen yang deficiency with damp group,with 10 rats in each group. Spleen yang deficiency model group were produced by Fanxieye irrigation stomach and subcutaneous injection of reserpine alternately and forced swimming rats composite method. Model of Spleen Yang misstating,giving external dampness group and humidification with Spleen Yang deficiency syndrome group moist environment for a week,were observed before and after making model and record the body weight of rats,eating,drinking,mental activity,hair gloss and stool and macro symptom score.2.After the completion of the damp environment,the spleen was removed,and the Con A induced lymphocyte proliferation was used to express the functional level of lymphocytes. To record and calculate the proliferation of spleen lymphocytes.3.After the completion of the damp environment,the spleen tissue was carried out by the conventional dehydration,paraffin embedding,embedding.5 slices were cut from each tissue block,and put in the 60℃temperature box.The 5 slices were stained with HE,and the pathological changes were observed under light microscope.4.After the completion of the damp environment,the spleen tissue 10mg/was removed,take the two-dimensional electrophoresis experiments, twodimensional electrophoresis image analysis,peptide mass fingerprinting and electrospray ionization tandem mass spectrometry.Results:1.Dampness on normal and spleen deficiency rats in general comparison. Damp environment of spleen deficiency rats compared with normal rats significantly reduced body weight,spleen yang deficiency plus damp group body weight of rats than damp group(321.70±18.74) decreased significantly,P<0.01. Spleen deficiency rats and external damp group in eating,drinking,mental state,hair color,stool and other relatively damp group changed.Spleen deficiency rats with damp group food intake(19.33±2.88)than damp group(23.83±2.93) decreased,P<0.05. Damp group(37.50±1.87),spleen yang deficiency rats with damp group water intake(36.67±4.18) compared with the control group(42.67±2.34) decreased significantly,P<0.01.Spleen yang deficiency and damp rats made die early emotional change gradually,showing irritability,quarrelsome state,put them in the swimming pool swimming,have rebounded. After modeling appears gradually in the doldrums,get together,lethargy,unresponsive,move slowly,hair withered and yellow,thin,soft faeces,shapeless,even dilute pond. Damp rats depilation is light,good mental state,occasionally sleepiness,grasping sounds more gentle.2.Damp on spleen lymphocyte proliferation of spleen deficiency rats and normal force comparison.In the damp environment,spleen yang deficiency rats and normal rats spleen lymphocyte proliferation were significantly lower than the control group.Compared with the control group,the spleen lymphocyte proliferation(47.94±1.13) compared to damp spleen lymphocyte proliferation(15.15±2.18),damp spleen yang deficiency and spleen lymphocyte proliferation(14.42±2.24) were significantly lower(P<0.01).Spleen deficiency plus damp group spleen lymphocyte proliferation capacity(14.42±2.24) than damp group lymphocyte proliferation capacity(15.15±2.18) no different P=0.476,P>0.05.With spleen deficiency plus damp group spleen lymphocyte proliferation capacity(14.42±2.24) compared with Spleen Yang deficiency syndrome group spleen lymphocyte proliferation capacity(36.07±3.05) significantly enhanced P<0.01.3.Damp on normal and spleen yang deficiency rats morphology of spleen tissue change.Damp group of spleen of rats white pulp marginal zone broadening,the white pulp occurrence center increases,spleen see light yellow pigment cell phagocytosis sonne.Rats of white pulp marginal band broadening was significantly higher than that of spleen yang deficiency and damp,splenic sinus was seen in phagocytosis shallow yellow pigment cells,spleen Sinus Dilation,congestion,spleen Sonne see pale yellow pigment cell phagocytosis.4.Damp on normal and spleen yang deficiency rats the differentially expressed protein spots in spleen tissue expression in comparison.Each experimental group selected three sample differential proteomic comparison,pairwise comparison between protein profiles and proteomic analysis,40 protein spots were identified,spleen yang deficiency and dampness group compared to the control group,the expression of protein 13 up,expression is down regulated in 20;damp group and the control group is up-regulated with 14,expression down regulated 19;damp group and spleen yang deficiency and dampness group is upregulated by 15,expression downregulation of 10.5.Damp of the differential proteins in spleen tissue and normal spleen yang deficiency rats were compared. In the experimental group 2-DE gel from expression of differential protein spots selected points clear and the expression level change 22 protein spots were clear as the final mass spectrometric identification of object and 20 proteins were identified by PMF method.Outside the damp environment in the normal rats were up-regulated and11,they are carrying fat apolipoprotein A-I,malate dehydrogenase,specific short chain acyl coenzyme A dehydrogenase,galactose 5,low molecular weight phosphotyrosine protein phosphatase LMW-PTP and anionic trypsin original-2 and intracellular chloride channel protein 1,F actin beta subunit,phosphoglycerate variable allozyme 1,film spike protein,actin related protein 2 / 3 complex subunit 5 and 4 genes down regulated expression of,they are 3- glyceraldehyde phosphate dehydrogenase,mammalian peroxidase 2,Ig kappa chain C region,B alleles,combined with globin.Outside the damp environment in rats with spleen deficiency upregulation of 9.They are carrying fat apolipoprotein A-I,malate dehydrogenase,specific short chain acyl coenzyme A dehydrogenase,galactose 5,low molecular weight phosphotyrosine protein phosphatase LMW-PTP and anionic trypsin original-2 and intracellular chloride channel protein 1,phosphoglycerate variable allozyme 1,moesin,expression down regulated 5. They are 3-glyceraldehyde phosphate dehydrogenase,mammalian peroxidase2,desmin,Ig kappa chain C region,B alleles,myosin regulation light chain 9.Conclusion:The control group and spleen yang deficiency rats. Simulation of damp environment factors were studied,including body weight,eating,drinking,mental state,activity,coat gloss,skin and mucosa,excrement,etc.and deficiency of Spleen Yang Group has more obvious effects.Simulated external damp environment factors significantly reduced the cell immune function in normal and Spleen Yang Group. External damp environment factors(external factors) relative to the deficiency of the spleen(internal factors) on the immune suppression of the body stronger. Factors(external factors) and spleen yang deficiency(internal cause) have synergistic effect on immune function of the body.The effect of external wet environmental factors on spleen tissue morphology of normal and spleen yang deficiency rats were affected,but the changes of spleen tissue morphology of spleen deficiency rats were obvious.There were different expression of protein in normal and spleen yang deficiency rat model. Simulated external damp environment factors and the role of spleen yang deficiency in the body of the difference between the protein has its specificity,there are intersection. These differences in protein function and cell structure,energy metabolism,cell apoptosis,cell proliferation and differentiation,signal transduction,calcium homeostasis,immune response,immune protection,tumor related. Under the action of different factors of specific proteins may is with Spleen Yang deficiency syndrome and dampness evil invasion related disease specific protein,and is likely to be the diagnosis of spleen yang deficiency molecular markers and damp pathogenic molecular markers. |
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