| Objective:1.To establish TaqMan-MGB Probe Real-Time Fluorescence Quantitative PCR to detect the concent of T-cell receptor excision DNA circles(TRECs)in peripheral blood.2.To investigate the clinical value of T cell receptor excision DNA circles(TRECs)and CD31+regulatory T cells in evaluating the recent thymic output function in patients with chronic hepatitis B.Methods:1.The primers and probes were designed with gene sequences of T-cell receptor(TCR).By establishing Taqman-MGB labeling probe in the quantitative determination of peripheral blood TRECs content through the repeatability,sensitivity,accuracy experiment evaluation of the reliability.2.105 patients with chronic hepatitis B and 30 healthy control patients were divided into four groups:mild chronic hepatitis B patients(Mild HBV,n=35),moderate chronic hepatitis B patients(Moderate CHB,n=35),severe chronic hepatitis B patients(Severe CHB,n=35)and healthy controls(HCs,n=30).Peripheral blood mononuclear cells(PBMC)were separated by density gradient centrifugation.CD31 was used as a cell surface marker of thymus recent output function.The proportion of CD31+Treg in Treg cells was detected by flow cytometry.The number of T cell receptor excision DNA circles(TRECs)in PBMC was detected by TaqMan-MGB double probe real-time fluorescence quantitative PCR.The correlation between CD31+Treg/Treg ratio and TRECs content in PBMC was analyzed by Spearman correlation test analysis.At the same time,CD3+,CD4+,CD8+,and CD4+/CD8+,ALT,HBV-DNA and hepatitis B immune markers were detected in three groups of patients with chronic hepatitis B.Results:1.TaqMan-MGB probe real-time fluorescence quantitative detection method for TRECs content was successfully established through many experiments.The minimum detection limit was 50 copies by detecting plasmid of 5×105 Copies in 10 times diluted solution.The mean of 6.18 Lg Copies/106BMCs and 3.54 Lg Copies/106BMCs which had tested 25 times for the same sample was 6.08±0.14,3.52±0.18Lg Copies/106BMCs,internal coefficient was2.26%,5.12%.The average of the 20 tested results was 6.10±0.23,3.51±0.22Lg Copies/106PBMCs,external coefficient was 3.85%,6.27%.For confirmed positive Ureaplasma urealyticum DNA,Chlamydia trachomatis DNA,herpesvirus type 2 DNA,gonococcal DNA,HBVDNA(UUDNA,CTDNA,HSV2 DNA,NGDNA,HBVDNA)in 5 cases respectively.No non-objective gene amplification was detected by double steaming water instead of template.2.The TRECs(3.53±0.66)LgCopies/106PBMC in patients with severe CHB was significantly lower than that(4.57±0.52)LgCopies/106PBMC of the mild CHB(p<0.001),and HCs group(4.82±0.75)LgCopies/106PBMC(p<0.001)in the four PBMC groups.The TRECs(4.08±0.58)LgCopies/106PBMC in the moderate CHB group was higher than the severe CHB group and lower than the mild CHB group;Although the number of TRECs in PBMC in mild CHB group was lower than that of HCs group,there was no statistically significant difference between the two groups(p>0.05).3.The percentage of CD31+Treg/Treg(12.25%±2.35%)in the severe CHB group with peripheral blood was significantly lower than the mild CHB group(16.35%±3.35%,p=0.002)and HCs group(17.75%±3.82%,p<0.001)in the four groups.That(14.38%%±2.77%)of in the moderate CHB group was between severe CHB group and mild CHB group,Although the percentage of CD31+ Treg/Treg of peripheral blood in mild CHB group was lower than that of HCs group,there was no significant difference between the two groups(p=0.098).4.The percentage of CD3+,CD4+,and CD8+T lymphocytes in the four groups was slightly different among the groups with peripheral blood and the proportion of CD4+T lymphocytes in the severe CHB group was the lowest.The proportion of CD8+T lymphocytes was the highest,and the performance of moderate CHB was the second,but the content of CD3+,CD4+,and CD8+ in the four groups had no statistical difference(p>0.05).The CD4+/CD8+ratio of moderate CHB was significantly lower than that of mild CHB and HCs,there was a statistical difference(p<0.05),but there was no statistical difference in the mild CHB group and HCs group(p>0.05).5.According to the ALT value,HBVDNA content and HBeAg,105 patients with chronic hepatitis B were divided into ALT<2ULN group and ALT≥ 2ULN group.The content of TRECs in ALT≥ 2ULN group was significantly lower than that of ALT<2 ULN group,there is a statistical difference(p<0.05).The percentage of CD31+Treg/Treg was also lower,and the two groups of data were the statistical difference(p<0.05).There was no statistical difference(p>0.05)in TRECs content and the percentage of CD31 Treg/Treg among the three groups of HBVDNA<102IU/ml,102≤HBVDNA<105IU/ml,HBVDNA≥105IU/ml.Similarly,there was no significant difference(p>0.05)in the HBeAg positive group and HBeAg negative group.6.The percentage of CD31+ Treg/Treg with peripheral blood was positively correlated with the number of TRECs in PBMC cells(r=0.551,p=0.014)in 105 specimens.Conclusions:1.TaqMan-MGB probe real-time fluorescence quantitative PCR was successfully established to detect the content of TRECs in peripheral blood.2.It is indicating that the recent thymic output function was reduced by the content of CD31+Treg/Treg and TRECs was reduced in patients with moderate and severe chronic hepatitis B.3.The content of CD31+Treg/Treg and TRECs are related to ALT level and no related to HBVDNA level,HBeAg positive or negative.4.CD31+ Treg/Treg is positively correlated with TRECs content and that is a new indicator for evaluating the recent thymus output function. |
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