Proteomic Research On Cultured Human Mesangial Cell Of IgA Nephropathy Patients Of Deficiency Of Both Qi And Yin

Objectives1. Empirical Study To establish the two dimensional gel electrophoresis maps of cultured human mesangial cell stimulated by serum IgAlfrom healthy people and IgA nephropathy patients of deficiency of both Qi and Yin, and to find the differential proteins, then to analyse them with mass spectrographic analysis, and to research them from Protein Date Bank to identificate them. To try to find the specific biomarker for exploring the non-traumatic diagnostic possibility of IgAN,and to select the possible target of drug for enhancing the clinic healing rate for IgAN treatment.2. Clinical Study To observe the relations between the symptom integration, experimental indexes, pathologic change with patho-grouping of IgA nephropathy, then to provide references for diagnosis,treatment and prognosis of IgA nephropathy.Methods1. Empirical Study1) Serum IgA1 was separated from normal subjects and patients with IgA nephropathy with Affinity chromatography and molecular sieve chromatography, and was identificated with Westernblot.2) Glomeruli were separated using a three-layer grit and digested with typeⅣcollagenase,then inoculated in culture flask in RMPI-1640. To detect the celluar markers including a-SMA, Vimentin, Cytokeratin and factorⅧof subage GMC with immunocytochemistry.3) The proteins of human mesangial cell stimulated by serum IgAlfrom healthy people and IgA nephropathy patients of deficiency of both Qi and Yin were separated by two dimensional gel electrophoresis. The gels were dyed with sliver staining and scanned for gel pictuers,to find out the differential proteins with software of ImageMaster5.0. The differential proteins were cut and digested with trypsin and identificated them with mass sepctrographic analysis, and to obtain the finger print map of peptide, then to search them through Protein Data Bank and identificate them.2. Clinical studies 70 cases of IgA nephropathy were divided into three different TCM syndromes.Firstly, deficient syndrome, including deficiency of both Qi and yin, spleen and kidney qi deficiency and hepatic and renal yin deficiency. Secondly, sthenia snydromes including water vaper,humid heat and pathogenic damp. Thirdly, asthenia and sthenia syndrome, including spleen and kidney qi deficiency with pathogenic damp and congestin. Their experimental indexes were collected and their symptoms were integrated.And 45 cases of IgA nephropathy renal needle biosy pathological reports and evaluate the pathological lesion by integrating according to Katafuchi IgA nephropathy integration standard. Relations of these indexes were analysed.Results1. Empirical Study1) Serum IgA1 was successfully separated from normal subjects and patients with IgA nephropathy with Affinity chromatography and molecular sieve chromatography, with high recovery rate and high purity.2) Primary cultured human mesangial cell could grow well, The immunocytochemistry stainings of a-SMA and vimentin were positive and those of factorⅧand cytokeratin negative in the cultured glomerular mesangial cells.3) The proteomics maps of cultured human mesangial cell stimulated by serum IgAlfrom healthy people and IgA nephropathy patients of deficiency of both Qi and Yin were initial established. The proteins of IgAN were (602±13) and the match rate was 95.97%; while the proteins of normal subjects were (569±5) and the match rate was 93.27%. There were twenty two proteins of IgAN exceeding three times over normal subjects. Besides only eight proteins expressed in IgAN and six proteins expressed in normal subjects. The fourteen proteins were identificated, eight of them had definite functions,which belong to heat shock protein, signal transducer, fibronectin, galactose and metabolic enzymes, and one of them was unknown protein.2. Clinical Study1) On age and sex, the difference of three groups are not statistically significant (P>0.05), but the pathogenesis of deficient syndrome and asthenia and sthenia syndrome was longer than sthenia syndrome, and the difference was statistically significant (P<0.05). 2) TCM pattern of snydrome:there was some correlation between deficient syndrome and asthenia and sthenia syndrome with nephridial tissue patho-ranking (P=0.032<0.05), and the patho-ranking of deficient syndrome and asthenia and sthenia syndrome mostly showedⅡ～Ⅳtype of Hass. the patho-ranking of sthenia syndrome mostly showed the type ofⅠ～Ⅱof Hass.3) TCM symptoms intergration and patho-ranking:The intergration is increasing with the patho-ranking augmentation, the difference among them was statistically significant (P<0.05).4) experimental indexes and patho-ranking:The experimental indexes of urine blood, urine protein,24 hours urine protein, BUN, blood creatinine, blood uric acid increased with the patho-ranking increasing, but only the difference of the 24 hours urine protein among them was statistically significant (P<0.05). The difference of other experimental indexes was not statistically significant (P>0.05). The change tendency of IgA, IgG, IgM, C3, C4, CRP, RF, ASO, ESR, TG, CHOL was not obvious with the patho-ranking increasing, The difference of them was not statistically significant (P>0.05).Conclusions1. Empirical StudyThere are some difference of proteins expressing on cultured human mesangial cell stimulated by serum IgAlfrom healthy people and IgA nephropathy patients of deficiency of both Qi and Yin, the different proteins may help reveal the pathogenesis, or discover the specific biomarker and find out the possible target of drug treatment of IgAN.2. Clinical Study1)TCM on IgA nephropathy:Firstly, the deficient syndrome, especially deficiency of both Qi and yin; secondly, asthenia and sthenia syndrome, especially spleen and kidney qi deficiency with humid heat,pathogenic damp and congestin,thirdly, sthenia syndrome.2) TCM symptoms intergration and pathological lesion intergration are increasing with the patho-ranking augmentation, and their correlation is positive.3) Experimental indexes and patho-ranking:only the difference of urine protein quantitation among them is statistically significant (P<0.05), which hint the urine protein is the independent risk factor of the prognosis of IgA nephropathy.