| Objective:1.To identify serum protein biomarkers in infants with hepatitis syndrome resulting from congenital human cytomegalovirus(HCMV) infection and intracellular and extracellular of HCMV infected glioma cells using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry(SELDI-TOF-MS).2.To apply immediate early protein into Tet-On cell line with regulatory system and to study its protection of injury and to supply new method and means in exploring HCMV IE protein anti-apoptisis and its possible mechanism.Materials and Methods:1.Serum samples were collected from 20 HCMV-infected infants with hepatitis and 25 controls.Of the 25 infants in the control group,5 were infected with HCMV but without hepatitis,10 had hepatitis but no HCMV infection,and 10 were healthy.Protcomic expression in the serum was detected by WCX2 chips and SELDI-TOF-MS.Differential protein analysis and identificaion by Biomarker Wizard software and Swiss Protein Database.2.Human glioma cell line(U251) and human neuroblastoma cell line(SH-SY5Y) mixed culture with cell population of 10 to 1 and after 6 hours,the level of HCMV infection was detected by HCMV IE.Using flow cytometer and SELDI- TOF-MS to detect differential expressionof intracellular and extracellular of HCMV infected cells.HCMV infection and the initial analysis and identification of related protein moleculars were performed by Swiss protein database.3.Specific primers were designed to amplify IE1 gene by pIE72 and to construct regulary eukaryotic expression vector pTRE2-hyg/IE1 before its purification and identification.4.Tet-On HeLa cell line was cultured with conventional culture.Cells were transfected with cation transfection reagent.Enhanced green fluorecentprotin report gene were transfected firstly to decide the transfection efficiency;Tet-On HeLa cells were culture in vitro and were transfected with pTRE2-hyg/IE1.After double screening by G418 and hygromycin B,IE1 expression was identified by RT-PCR and Western Blot,as well as immunohistochemistry.5.HeLa cell line was induced into apoptosis by the final concentration of TNF-alpha(100 ng/mL) and thus the HeLa cell apoptosis curve was set up;Different final concentration Doxcycline(0.1μg/mL,1μg/mL and 10μg/mL) acted transfected pTRE2-hyg/IE1 HeLa cell line to induce IE1 expression.RT-PCR and western blot were used to detect the expression of IE1 gene and protein,respectively.MTT method was used to assay the increasement of HeLa cells.Results:1.Comparing with control groups,fifteen protein peaks were distinctly different among the four groups in the mass range from 2,000 to 20,000 Da.Of these 15 peaks,four at 4,349.8, 5,808.7,7,935.6 and 8,885.9 Da were significantly different between the congenital HCMV-infected infants with hepatitis and the controls.Five peaks were distinctly up-regulated in the infants with HCMV infection(3,266.8,5,638.5,5,909.1,7,771.4 and 15,835.6 Da) compared to those in infants without HCMV infection.Two proteins at 4,600.1 and 5,704.3 were up-regulated in infants with HMCV infection but no hepatitis. Four protein peaks were markedly different(7,567.0,13,744.8,15,100.7 and 15,915.0 Da) between the infants with hepatitis and the other controls.2.Conmparted with non-infected group,the two HCMV infected infants' sera show five protein with marked changes,among which peaks at 5639.0Da,5909.6Da,7776.5Da and 15833.2Da were very close with TP,beta-amyloid precusor A4,platelet factor 4 and IL-25.3.Compared with other groups,HCMV inapparent infection group shows two changes, among which the protein peak at 5710.7 Da was close with beta-defensin.4.Compared with normal hepatic function group of infants,four proteins in serum show marked changes,among which the peaks at 7567.6Da,13744.1Da,15092.8Da and 15931.6Da were very close with MIP4,PA,ALR and Hp. 5.RT-PCR was applied to detect HCMV IE gene expression to confirm the infection of HCMV and the HCMV-infected-neuroma cells model was constructed successfully.No clear changes were found in the 1 day of infection while in the 3 day of infection,the G1 peak left side showed hypodiploid cell pear which appeared more marked apoptosis in the 5th day of infection.6.SH-SY5Y cells showed apoptosis after infection and according to the infection time,the apoptosis increased.The apoptotic rates were 4.1%and 42.6%.Comparing with non-infected group,the protein peaks at 2631.6Da,12027Da and 13536.3Da up-regulated after HCMV infection.At 48 hours,the inceasing was more obvious than that of at 4 hours.Searching in Swiss data base found that they were very close with Capspase-1, TNF-alpha and beta-amyloid precusor protein.7.Eukaryotic expression vector pTRE2-hyg/IE1 was constructed successfully with sequencing.A stable expression HeLa cell line was set up with Dox induction named pTRE2-hyg/IE1 HeLa cell line after G418 and hygromycin B.Gradient concentration (0.1μg/mL,1μg/mL and 10μg/mL) can induced pTRE2-hyg/IE1-HeLa for 48h, mRNA was detected by RT-PCR with results of IE1/β-actin were 0.733,0.917 and 1.768,while IE1 proteins were detected by western blot with results of IE1/β-actin were 1.32,5.83 and 7.07.8.TNF-a and ACTD with final concentrations of 100ng/mL and 25ng/mL respectively were combinded and added onto HeLa cells transfected by IE1 plasmind.Activity of HeLa showed that in 8 hours of infection,transfected IE1 HeLa cell group activity have marked difference comparing with the non-transfected group.Caspase-3 expressoin was detected by CaspACE kit as 0.6±0.029,1.6±0.041 and 1.85±0.065,while non-infected HeLa cells,the caspase-3 expression were 0.85±0.061,2.6±0.058 and 4.5±0.065. Significance of difference in statistics analysis were shown with p＜0.01.All the data proved that IE1-HeLa have functions of anti-apoptosis with confirm of cytology.Conclusion:1.Protein database in sera and intra-extra cellular proteins in both hepatitis syndrome resulting from congenital HCMV infection and HCMV infected neuroma cells was constructed and proteomics indexes were found in HCMV infection individuals and cytology changes.2.Up-regulated cell protein factors in procession of HCMV infection may play certain role in apoptosis.3.Regulatory Tet-On plasmid could produce dose-dependent interesting protein and play cytological functions by the induction of Dox. |
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