Analysis Of Proteomics In Rat Corneal Transplantation Rejection By Surface Enhanced Laser Desorption/Ionization-Time Of Flight Mass Spectrometry

Background:Graft rejection is the main reason lead to the failure of cornea transplantation. Especially on the high risk cornea transplantation patients with great number new vessals on the recipient bed,graft rejection is serious and always begins early. Therefore it reduces great destruction to the graft.With immunosuppressive agent's regionally or systemic use,cornea implant's survival rate is still less than 35%and this is a problem need to be resolved urgently.Graft rejection is a complex process effected by multiple factors,which are widly separated and overlapped.Nowadays,cornea's basic research mostly stays in single gene and genome level.However,real physiology function's executant in vivo is protein.Reports published already concerning mRNA level and protein expression peak are not completely matched in cell,thus study disease in protein level has become very important.However,traditional method researches signal or several proteins can not express the basic mechanism of cornea transplantation rejection systemicly and clearly.In order to get a full-scale and penetrating understanding of the complex process of graft rejection,we must research protein in total,developmental and associated.Reports have indicated that some drugs' systemic effects are much better than the regional effect in the treatment of cornea transplantation rejection.This reminds us to observe the cornea transplantation rejection in total:concerning the nutrition resource of cornea and blood circulation's effect.Cornea has no blood vessels.It's nutrition sources are the limbus vessels,aqueous,and tear.epithelial cells nutriated by tear while endothelial cells nutriate by aqueous.Energy,which mainly comes from glucose,is obtained mostly by endothelial cells through aqueous. The others come from tear and limbal vascular supply.Therefore,we can get the conclusion:the status of the cornea changes its own fate when tear,aqueous and blood components change.At the same time,to combination of clinical practice, collect tears,aqueous and serum are safer and more easy compared with cornea collection.Proteome is a protein group coded by a genome.Proteomics research the whole proteins expressed by a genome,organism,cell or tissue and the interactivity among them.which can supply data reflect gene,tissue or organ's statement more veritably.SKLDI-TOF-MS(surface-enhanced laser desorption/ionization time of flight mass spectrometry) is a combination of protein chip and surface enhanced laser desorption ionization-time of flight mass spectrometry.The advantage of SELDI -TOF-MS include small amount of sample,high sensitivity,high-flux and directly test the un-purified samples.Corneal proteomics is still at the very beginning in world wide.Many of tear protein components are excrime proteins.All of them can be effected by both regionally or systemic factors.It has found that there are characteristics of the tear protein composition changes while the body is in fail.A great extent of low abundance proteins in tear play important role in physiology and pathologic.It is reported that the extent of IgG,sIgA,LF changed regularity after keratoplasty.When the rejection occurred,it can be find that level of IgG increased steeply and the level of LF declines.To test the content of IG in tear can prevent the rejection and monitor if it is cured.Another research indicates that albumin,H-type cytokeratin andα-1 antitrypsin elements are significantly elevated in acute rejection stage.This result suggests that there are at least three mechanisms of the change of aqueous liquid elements:the broke of aqueous-blood barrier,enzyme degradation and protein regionally composite release into aqueous.However,there is no report about the proteome change and its analysis in serum.Purpose1.Improve rat cornea transplantation model building technique on the basement of original model and discuss the mechanism of cornea transplantation immunological rejection on histomorphology aspect.2.Test the difference of protein composition in tear,aqueous liquid and serum between cornea transplantation immunological rejection rats and rats with no rejection by SELDI protein chip technology.Find the specific protein fingerprint and validate the specificity and sensitivity of the model.Flit cornea transplantation immunological rejection associated protein with comparison and analysis of the change in protein elements,then deduce the possible mechanisms of cornea transplantation immunological rejection.Methods1.Build homogeneity penetrability corneal transplantation rat model:20 Wistar rats as donors and 40 SD rats as acceptors with penetrability corneal transplantation on the right eyes of SD rats.The grafts of groupⅠcome from side-center and the suture tail remained about 1 mm while grafts of groupⅡcome from center cornea and the suture remained as short as possible.2.Divide the rats into two groups randomly,rejection groupⅠ(saline control group) and rejection groupⅡ(CsA and TobraDex),20 ones each group.Observe with slit lamp microscopy from the 1^st day after operation and score the cornea in opacity,edema and new vessels three indexes.The result datas as means±SD and test by Independent-Sample T Test in SPSS13.0 software.The difference is significant while P＜0.05.3.Get 2 corneas from each group randomly on the 7^th day after operation, observe with HE dying and routine transmission electron microscopy.4.Meanwhile,gather blood,tear fluid and aqueous fluid from other rats. Standing the blood on ice for 1h after gathering,centrifugate on 0℃.5000r/min 10 min.Separate the serum and frozen in -80℃together with tear fluid and aqueous fluid.for the next SELDI-TOF-MS.5.Divide the 18 tear,aqueous and serum samples into 2 groups randomly,9 samples each group to establish the decision tree model with one group:capture protein with WCX2 protein chip,when the proteins are ionized,test the tear,aqueous and serum samples on each group with PBS IIC type protein reading machine and get protein fingerprint from each sample.Automaticly gather data with Ciphergen ProteinChip 3.0 analyze software,Ciphergen Biosystems Std,build decision tree model with the albumin tested by albumin chip based on min Gini index method with Biomarker Wizard software,than estimate with decision tree model with 10-fold Cross-validation Estimates,find out the protein combination that can best distinguish the two group samples.6.Then test model's sensitivity and specificity with another sample group blindly. 7.Preliminary screening the marked proteins in Swiss-Prot and TrEMBL protein library.Results1.Both two groups have expected progresses and there have no effect on the cornea and body after collecting samples.2.According to homogeneity penetrability cornea transplantation score,we can definite that rats in rejection groupⅠhave already got cornea transplantation rejection on the 7^th day after operation,whike rats in rejection groupⅡdo not.3.HE dying shows vacuoluses formed by stroma cell of epithelial lamina,raritas interstitium,many inflammatory cellinfiltrate that lie in epithelial and superficial stroma,new vessels lie in stroma.4.TEM shows the epithelium of rejection grafts are disordered,abscure in fine structure, fibrous in cell bridges and lost the microvilli of surface cell.The tunica propria of rejection grafts lost it's regulariy parallel arrange with the condensed nucleus,margining chromatin,uncleared caryotheca,obscure fine sturcture and dilat reticulum whie it's collogen got cutinization.We can find glucogen in both epithelium and stroma and lipid droplet in stroma.5.In the range of molecular weight 1000—20000 Da:5.1 Tear5.1.1.108 proteins are marked in total in tear sample of rejection and non-rejection group,there are 11 kinds of proteins which have classify value,among these 6 kinds got high classify value,M2167.66,M3356.53 are down regulated in rejection group while the M1169.86,M1183.69,M9238.59,M11882.2 are up regulated.5.1.2.The result of blind detection,the sensitivity of this decision tree model is 78%and the specificity is 89%. 5.1.3.The result of proteins search library result suggest that the down regulatin related proteins maybe Pro-histogranin and Glucagon-like peptide 1;while the up regulatin proteins maybeβ-defensins 2,caspase-3 subunit,and neurokinin A.5.2 Aqueous5.2.1.87 proteins are marked in total in aqueous rejection and non-rejection group,there are 6 kinds of albumin which have classify value,all of these 6 kinds got high classify value,M1037.98,M1436.29,M1326.12,M1082.37,M1067.86, M11261.6 are up regulated.5.2.2.The result of blind detection,the sensitivity of this decision tree model is 67%and the specificity is 78%.5.2.3.The result of protein search library result suggest that the related albumin maybe Angiotensin-1,Angiotensin-2,Neuropeptide-glutamic acid-isoleucine.Transforming growth factor beta-2.5.3 Serum5.3.1.58 proteins are marked in total in tear rejection and non-rejection group, there are 6 kinds of proteins which have classify value,all of these 6 kinds got high classify value,M8809.89,M7010.01,M4420.15,M3507.18,M4151.08 and M1706.30 are up regulated.5.3.2.The result of blind detection,the sensitivity of this decision tree model is 75%and the specificity is 75%.5.3.3.The result of albumin search library result suggest that the related albumin maybe Neuropeptide Y,β-defensin 1,Glucagon,and Cortistatin-14.Conclutions1.According to the technique improvement in building rat cornea transplantation model and discussion of sample collection method,arrange an effective operating instruction to build models and collect samples.The result of keratoplastia can be effected by remaining longer suture and selecting the posion of cornea implant.2.The 7^th day post-operation is sample collection time.It is the time to distinguish the graft rejection group from non-rejection group.3.clinical observation Evaluation after rat cornea transplantation is correspond with HE dying histomorphology observation result,4.The result of transmission electron microscope shows that there are both cell apoptosis and necrosis in rejection graft.So can we conclude that cell apoptosis plays role in the graft rejection.5.It is reliable to obtain proteomic profiles in tear,aqueous and serum for rat marker of cornea graft rejection using SELDI-TOF-MS with well reproducibility.The biological mark in tear,aqueous and serum which detect by SELDI-TOF-MS technology can correctly distinguish rejection and non-rejection rats..6.We can assume that all of them take part into the rejection of corneal thansplantation by the means that the pro-histogranin transform into histogranin,β-defensins stimulate the activation of immunity directly and indirectly,and CST stimulate the monocytic differentiating and activating.7.The up-regulation of neuropeptide in tear,aquous and serum suggests that maybe neuripeptide play an important role in graft rejection by correlate with monocytes differentiation and activation.It makes us think about the position of the nervous system in graft rejection.Innovation point1.To normalize the technique of constructing openetrating keratoplasty rat model and the agendum of collecting samples firstly. 2.To obtain proteomic profiles in tear,aqueous and serum for rat marker of cornea graft rejection using SELDI-TOF-MS first time.3.Proposed for the first time that neuripeptide may play an important role in graft rejection by correlate with monocytes differentiation and activation.