| Objective:Detect the percentages of Th17 and Treg which occupied the CD4+T lymphocytes and the relative mRNA expression level of the specific transcripton factors of each cell——RORγt and Foxp3 at the time of pre-treatment and after-treatment.Accompanied with dynamic detection of the associated indicators which are used to evaluate the level of ALT,AT,TBIL,HBeAg and HBV DNA.Thus,to discuss the changes of Th17 and Treg as well as their specific transcripton factors——RORγt and Foxp3 during the antiviral course of ETV and the effect of the balance between Th17 and Treg during the process of CHB.Methods:Sixty patients diagnosed with HBeAg(+)CHB who visited the first affiliated hospital of Kunming medical university during the course between May,2015 and August,2016 were enrolled in this study,among them,there are 38 male patients and 22 female patients.At the same time,choose 22 health volunteers from the medical examinational center,among them,there are 14 male volunteers and 8 female volunteers.All the CHB patients took ETV 0.5mg/d as the antiviral treatment.Detect ALT,AST,TBIL,HBeAg and HBV DNA at the time of pre-treatment of ETV,after 12 weeks,after 24 weeks and after 48 weeks.Detect the percentages of Th17 and Treg as well as the relative mRNA expression level of their specific transcripton factors——RORγt and Foxp3 at the time of pre-treatment of ETV and after 48 weeks.HBV DNA was dectected by qPCR equipment.Indicators which reflect liver function contain ALT,AST and TBIL were detected by automatic biochemical analyzer.The percentages of Th17 and Treg were detected by flow cytometer,while the relative mRNA expression level of their specific transcription factors were detected by RT-qPCR equipment.HBeAg was detected by ELISA.Statistical analysis of all collected datas were carried out by SPSS 19.0 version.Results:1.Male patients and female patients occupy 63.3%(38/60)and 36.7%(22/60)of HBeAg(+)CHB patients respectively,thier mean age is 35.8±7.3.The average level of HBV DNA is 1.83×108 cp/ml.The baseline level of HBV DNA is above 1.0×105 cp/ml in all of the CHB patients and among them,81.7%(49/60)of the CHB patients,their HBV DNA baseline level is above 1.0×105 cp/ml.Compare CHB patients with the health volunteers at the time of pre-treament of ETV,the level of ALT(211.60±103.11 vs 27.64±6.16),AST(143.57±65.94 vs 22.41±4.40),TB(15.69±5.63 vs 12.24±2.36),Th17(3.18±5.80 vs 3.19±1.36,Treg(3.25±1.44 vs 1.60±0.91),RORγt(2.77±2.31 vs 1.41±1.08)and Foxp3(2.75±1.14 vs 1.98±0.80)in the peripheral blood of CHB patients are significantly higher than the health voluteers(P<0.05).Compare CHB patients with the health volunteers at the time of after treament of ETV for 48 weeks,there is a significance(P<0.05)among the level of ALT(34.25±6.71 vs 27.64±6.16),AST(27.50±4.00 vs 22.41±4.40)and RORyt(1.70±0.88 vs 1.41±1.08)in peripheral blood.While there is no significance among the level of TB(12.82±3.71 vs 12.24±2.36),Th17(2.96±1.67 vs 3.19±1.36),Treg(1.88±1.12 vs 1.60±0.91)and Foxp3(1.62±0.99 vs 1.98±0.80)in peripheral blood(P>0.05).2.There is a significant decrease(P<0.05)among the level of ALT(39.00±21.50;36.00±7.00;34.25±6.71 vs 211.60±103.11),AST(32.00±18.25;29.50±6.75;27.50±4.00 vs 143.57±65.94),TB(15.41±4.20;13.29±3.74;12.82±3.71 vs 15.69±5.63)and lgHBV DNA(4.31±2.15;3.09±1.44;3.00±0.00 vs 7.66±0.91)in peripheral blood at the time of after treatment of ETVfor 12 weeks,24 weeks and 48 weeks when compared with the time of pre-treatment of ETV,except for compare TB between the time of pre-treatment of ETVand after treatment for 12 weeks.At the time of treatment for 12 weeks,24 weeks and 48 weeks,the recurrence rate of ALT is 53.3%(32/60),78.3%(47/60)and 93.3%(56/60)respectively,the negative conversion rate of HBV DNA is 31.7%(19/60),48.3%(29/60)and 80.0%(48/60)respectively,the negative conversion rate of HBeAg is 15.0%(9/60),20.0%(12/60)and 31.7%(19/60)respectively.3.When compared with pre-treatment of ETV,either the percentages of Th17(2.96±1.67 vs 3.18±5.80)and Treg(1.88±1.12 vs 3.25±1.44)which occupied the CD4+T cells in peripheral blood nor the relative expression level of their specific transcription factors——RORyt(1.90±1.01 vs 2.77±2.31)and Foxp3(1.70±0.88 vs 2.75±1.14)at the time of treatment for 48 weeks,except for RORyt,all the other indicators decrease significantly(P<0.05).At the time of treatment for 48 weeks,compared Th17(2.29±1.08 vs 5.63±3.67),Treg(1.52±0.90 vs 3.35±0.59),RORyt(1.63±0.91 vs 3.01±0.54)and Foxp3(1.49±0.82 vs 2.56±0.52)of the patients who had no HBV DNA negative conversion(12/60)with the patients who had HBV DNA negative conversion(48/60),all of them decrease significantly(P<0.05).It is same when comparing Th17(3.4211.84 vs 1.97±1.26),Treg(2.32±0.98 vs 0.95±0.77),RORyt(2.25±0.86 vs 1.15±0.93)and Foxp3(2.01±0.75 vs 1.04±0.77)between the patients who had no HBeAg negative conversion(19/60)with the patients who had HBeAg negative conversion(41/60).4.The ratio of Th17 and Treg(Thl7/Treg)of CHB patients is significantly decrease when compared with health volunteer(1.56 ±0.55 vs 2.92 ±2.41,P<0.05)at the time of pre-treatment of ETV.There is no significance when compare Th17/Treg between CHB patients and health volunteers(1.62±0.99 vs 2.92±2.41,P>0.05)at the time of after treatment for 48 weeks.Also,there is no significance of Th17/Treg between the time of pre-treatment and the time of after treatment for 48 weeks(1.56±0.55 vs 1.62±0.99,P>0.05).5.Analysis via GLMM and rank correlation reveals that during the course of ETV’s antiviral treatment,the influences of HBV viral load on the percentages of Th17 or Treg which occupied the CD4+T lymphocytes as well as the relative mRNA expression level of their specific transcription factors——RORγt and Foxp3 are more important than the influences of the other 3 indicators(ALT,AST and TB).Besides,there is a positive correlation between the percentages of Th17 or Treg as well as the relative expression level of their specific transcription factors——RORγt and Foxp3 and HBV DNA(r=0.627,0.704,0.721 or 0.657;P<0.05).Furthermore,HBV DNA contributes more to the percentage of Treg when compared with the percentage of Thl7.6.Either the time of pre-treatment of ETV or 48 weeks after treatment of ETV,there is a significantly positive correlation between the percentage of Th 17 and the relative mRNA expression level of its specific transcription factor——RORγt(r=0.845,P<0.05)as same as correlation between the percentage of Treg and the relative mRNA expression level of its specific transcription factor——Foxp3(r=0.890,P<0.05).Conclusions:At the time of active replication of HBV DNA which the same as the time of pre-treatment of ETV,Thl7 plays the role of pro-inflammatory while Treg plays the role of anti-inflammatory,thus for the CHB patients who hadn’t have antiviral therapy,the human body would make the CD4+T lympocytes differentiate into Treg through the immunoregulation by itself so that protect the liver from excessive inflammation.When the patients start taking the antiviral medicines——ETV,the differentiation from CD4+T lympocytes to Th17 or Treg were both blocked.It indicates that ETV not only inhibits the replication HBV,but also takes part in immunomodulating the inflammatory course which triggered by immune system.That’s why the control of liver inflammation was speeding up and HBV DNA can be an indirect indicator to the immunoregulation.Furthermore,the balance between Th17 and Treg incline more to Treg at the time of active replication of HBV DNA and thus to control the hepatic inflammation.With the progress of ETV antiviral therapy,the balance between Th17 and Treg recover and it is meaningful to control the inflammation of liver——protecting the liver inflammation from progressing into the liver fibrosis or even the LF.So the ratio between Th17 and Treg can be a meaningful indicator to predict the prognosis of CHB patients. |
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