Study On The Relationship Between Serum Levels Of Th1/Th2 Cell Related Cytokines And The Results Of Interferon Therapy In Patients With Chronic Hepatitis C

ObjectiveTo investigated the changes of Th1/Th2 cell related cytokine levels in serum in patients with chronic hepatitis C and the relationship between these cytokines and the outcome of interferon therapy.Materials and MethodMaterials1,Study objectThirty patients with chronic hepatitis C were enrolled in the present study(15 males,15 females;mean age 48.5±8.11years).All were treated with a 48-week course of IFN alpha-2b plus ribavirin.Peg-IFN-α-2b were injected via an intramuscular at the dose of 50μg once a week in eight patients,other twenty-two patients used IFN-α-2b at the dose of three million units(MU) three times a week.During IFN therapy,ribavirin was given orally at a dose of 800mg or 1000mg a day according to the weight(800mg for those weight＜60kg and 1000mg for those weight＞60kg).Those patients who were HCV-RNA negative and with normal ALT at the 24th week after completion of the IFN alpha-2b plus ribavirin combination therapy were declared as response group.Fifteen healthy sera were obtained from medical examination center of Shengjing affiliated Hospital of China Medical University as normal control.2,Main reagents and apparatusHCVRNA extraction reagents:TRIZOL,chloroform,isopropyl alcohol,75% ethanol,DEPC water etc;Purified reagents of sequencing product:EDTA(PH8.0), NaAc(PH5.2),anhydrous ethanol,70%ethanol,HiDiFormamide(formamide)etc;2% Agarose,TBE,RT-PCR amplification kit,PCR purification kits and PCR reaction primers are purchased from TaKaRa(Dalian) company limted.;cytokines of IL-2,IFN -γ,IL-6,IL-10,IL-12p40 and IL-12p70 detection kits are purchased from the Beijing-jingmei Biological Engineering company limted.IL-5 detection kit is purchased from Shanghai Senxiong Technology Industrial company limted.;PCR amplification instrument:PE USA incorporated products,pattern number is PE2400.; Bio-Rad,Beijing six-one instrument factory;Gel Imaging System,SHIDZU corpation; UV spectrophotometer,model UV-160A,USA;ABI 3130 automated sequencer:ABI the United States;XW-80A vortex oscillator:Shanghai Qingpu Luzhou West Instrument Factory K10CD;Dry thermostat:Hangzhou Blue Flame Technology Limted.;Low self-balancing centrifuge:LD25-2A,Beijing Medical Centrifuge Factory;Desktop high-speed centrifuges:Shanghai Medical Instrument Factory productionanalysis,model TGL-16G;Bole 680 automatic microplate reader,BIO-RAD American companies;Ultra-low temperature refrigerators,MDF-1155-1520C,Sanyo company of Japan;cell constant temperature incubator,WJ-22C Mar.NO.W-1343, Japan.Methods1,Detection of serum cytokinesSerum IL-2,IFN-γ,IL-5,IL-6,IL-12p40 and IL-12p70 were dected by ELISA according to kit's instruction.2,Detection of HCV GenotypeDetection of HCV Genotype used the method of direct sequencing.Genotype was classified according to the nucleotide sequences of 5'UTR region.HCV RNA was extracted from serum samples,reverse transcript to cDNA,amplification 5'UTR region fragment using PCR,which is 450 bp.The PCR products are purified and sequenced in the central laboratory of Shenyang Contagious Disease Hospital.At last the nucleotide sequences were analyzed in NCBI net to make sure the HCV genotype.3,Detection of HCVRNA loadThe HCVRNA load in serum was detected in fluorescence quantitative PCR according to the kit's instruction in Central Laboratory of Shengjing Hospital of China Medical University.4,Detection of leveles of serum ALT The serum ALT level was detected using automatic biochemistry analysis 8200A by Central Laboratory of Shengjing Hospital of China Medical University.5,Statistical analysisStatistical analysis was performed with the aid of the software SPSS 15.0.The analysis methods included the comparation of average,x~2 analysis and Biraviate dependablity analysis.P values＜0.05 were considered statistically significant.Results1,The outcome of interferon therapyThe response rate of the patients who were treated with the IFN plus ribavirin were 46.7%.30 patients were finally divided into 14 complete responders(CR group) and 16 nonresponders(NR group) at 6 months after the end of IFN therapy.Serum ALT levels were significantly higher in the CR group,almost twice as much as those in the NR group.Whereas,pretreatment viral load was not significantly different between the two group.2,The distribution of HCV genotype and the response rate of interferon therapy among patients of different genotypeGenotype 1b was more frequent in the NR group.Other parameters including gender,age,weight and time of infection were not different between the two groups. Patients treated with Peg-IFN-α-2b showed higher SVR rate(62.5%) compared with those with IFN-α-2b(40.9%),but statistical analysis was no significance.3,Changes of serum cytokine levels of patients with chronic hepatitis CThe serum level of IL-2 in chronic hepatitis C patients was significantly lower than that in the healthy control group.The serum levels of IL-5 and IL-12p40 are significantly higher than the latter.The serum levels of other cytokines had no significant difference between them.4,The relationship between cytokine levels in serum and HCV genotypeThe serum level of IL-6 in HCV genotype 1 patients was significantly higher than that in genotype 2 patients,the other cytokine levels had no differences between two genotypes;However the cytokine levels between genotype 1 and 3 had no significantly difference and the same as between genotype 2 and 3.Comparison with all subtypes: the level of IL-2 in 2a is significantly lower than that in 2b.5,The relationship between cytokine levels in serum and serum levels of ALT and HCVRNA loadSerum levels of IL-6 were directly proportional to the serum levels of ALT,while inversely proportional to that of HCV RNA load.The cytokine levels had no significant correlation with ALT and HCVRNA load.6,The relationship between serum cytokine levels and the outcome of interferon therapyThere were no difference of all cytokines detected between the groups of response and nonresponse before IFN therapy,but the level of IFN-γwere increased after interferon therapy in the response group.Conlusion1.There is Th1/Th2 cytokones imbalance in chronic hepatitis C patients.The decreased Th1 cytokine levels and increased Th2 cytokine levels in serum may be associated with hepatitis C chronicity.2.IL-6 is posbilily associated with liver inflammation.3.The level IL-6 of HCV genotype 1 patients was higher than that of genotype 2, And IL-2 level in genotype 2a was lower than that in 2b.Other cytokines detected had no relationship with the HCV genotype.4.There were no correlation between the levels of Th1/Th2 cytokines in the serum before IFN treatment and the outcome of IFN therapy.Increasing IFN-γin the serum induced by IFN treatment is associated with sustained response.