Applying ClinProt Technology To Detect Relevant Proteins In Normals And Psoriatic With Different Syndrome Patterns

ObjectiveTo detect plasma proteins and T Lymphocytes membrane proteins in normals and psoriasis patients with different syndrome patterns by using ClinProt combine MALDI-TOF technologies, as a result to find out the difference of protein expression between normal population and psoriasis patients with different syndrome patterns. This study tried to connect T lymphocyte membrane protein receptor to plasma protein ligand from the longitudinal level. Reveal the scientific meaning of Chinese medicine syndrome pattern, and to appraise the fact of "zheng" in a holistic concept. Hence, to provide for substance basis for syndrome differentiation and breakthrough in Traditional Chinese Medicine(TCM) modernization.Methods1.Subjects:(1)Experiment group:A total of30cases were diagnosed with psoriasis vulgaris, and were allocated into blood-heat syndrome, bloodstasis syndrome, blood-dryness syndrome groups, with10cases in each group, according to the inclusion criteria.(2)Control group:10healthy individuals who matched the psoriasis group both in gender and age were selected as control group.2.The experimental method:(1)Collected plasma:Extracted blood samples10ml with anticoagulant tube containing heparin, shaked, centrifuged, supernatant plasma was taken at1ml and was preserved at4℃.(2)Extracted T lymphocyte membrane protein:First, T lymphocytes in peripheral blood:Residual plasma was shook before adding equal amounts of phosphate buffer solution(PBS). Extracted mononuclear cells from the peripheral blood according to the operation of Lymphocyte Separation Medium. During extraction, plasma should be washed by PBS repeatedly in order to remove impurities like platelet thoroughly. Second, Extracted T lymphocytes though Magnetic cell sorting According to Pan T Cell Isolation Kit: preparation for sample; magnetic marker; magnetic cell sorting with LS column. Plasma and T lymphocytes were preserved at4℃. Third, Extracted the T lymphocyte membrane protein:Added the homogenization buffer and enzyme inhibitor according to kit instructions, repeated grinding cells until more than70%cells were splited under the microscope when observed. T lymphocyte membrane protein were obtained by gradient centrifugation.(3)Establishment of proteinic spectrum with plasma and T lymphocytes membrane protein:Preparation and quantitation of protein sample. Prepared samples, standard, matrix according to the kit. Mixed the sample, standard respectively with matrix and blended into crystals. Took the crystals into the mass spectrometer for mass spectrometry analysis and searched spectrums.3.Statistical analysis:Flexcontorl3.3.85.0was used for data collection with a linear model: LP-clinprot at voltage of25kv, CPS standard system was used. The measurement ranged from Okd to lOKd, frequency of laser was100Hz and intensity was40-60. Triggered1000. Software of standard peak was flexanalysis3.3.65.0. Then imported the sample spectrum into the statistical analysis software of ClinProTools2.2for analyzing, choosing different peaks according the standard to P<0.05, ratio>1.5.ResultsWhen comparing to the healthy controls, some differently expressed protein peaks were identified in the psoriasis with different syndrome patterns of T lymphocytes membrane protein. One of blood-heat was up (4747Da). One of bloodstasis was up(4747Da) and two of them were down(2068Da,7764Da). One of blood-dryness was up (4747Da). In addition, there were also significant differences in the protein peaks of plasma in the psoriasis with different syndrome patterns when compared with healthy controls. Three of blood-heat were up (6631Da,9288Da,4965Da). Five of bloodstasis were up (2863Da,4418Da,6631Da,4965Da,3241Da). Two of blood-dryness were up (4965Da,9288Da), One of which was down(3957Da).Conclusion 1. This was the first time using ClinProt technique to detect differential protein peaks compared patients with different syndrome patterns of psoriasis vulgaris to healthy people in this study, and differential protein peaks were detected in T lymphocyte membrane and plasma. Different syndromes had differential protein peaks, which suggesting these proteins might be related to the pathogenesis of psoriasis with different syndrome patterns.2. Compared with healthy people, blood stasis syndrome of psoriatic detected more differential proteins in quantity and greater differences, suggesting that blood stasis may be the key involved in the pathogenesis of psoriasis.3. Since the experiment was only detected molecular weight of differential proteins in normals and psoriatic with different syndromes, but faild to identify a specific protein. Therefore it could not have clear differences between psoriasis with the same syndrome pattern in T lymphocyte membrane proteins and plasma proteins. Look forward to increase the amount of samples, more quantities and specific protein peaks may be extracted to identify for farther research.