Expression Of Perforin In The Peripheral Blood HBV-specific CTL、NK And NKT Cells Of Patients With Chronic HBV Infection

Backgroud：There are about93million people infected HBV in China. HBV cancause different clinical outcomes,including acute hepatitis B、chronic HBV carriers、chronic hepatitis B、liver failure、cirrhosis and hepatocellular carcinoma.The main reasonis determined by the immune response status.The human liver contains a lot of innatecells.such as NK cells(natural killer cells, NK)、 NKT cells (natural killer Tlymphocytes,NKT),accounting for2/3of the whole liver lymphocytes.About30percent isNK cells,NKT cells are also nearly30percent, while the adaptive immune T lymphocytesubsets account for only about30%.Perforin(perforin,also as pore-forming protein,PFP)issecreted by cytotoxic cell such as NK cells、NKT cells and cytotoxic T lymphocytes(cytotoxic T lymphocytes CTL)effector molecules.From a transmembrane pore in thetarget cells,perforin makes granzyme into the target cell cytoplasm,which initiate apoptosissignaling pathway,causing liver cell damage.Therefore, the role of NK cells、NKT cells andCTL in the clearance of HBV infection and immune which causes pathological damageneed to be further clarified.Objective: To study the expression of perforin (also known as pore-forming protein,PFP) in the peripheral blood HBV-specific CTL、NK and NKT cells of patients withchronic HBV infection and to analyze its relationship with serum alanine aminotransferaselevels、HBV viral loads. Investigate the immunological characteristics of chronic HBVinfection patients in peripheral blood with HBV-specific CTL、NK、NKT cells and theirmechanism mediated liver injury.Methods：The subjects were the HBV infection patients from outpatient or inpatient from September2011to March2012, First Affiliated Hospital of SuZhou University.15cases of hepatitis B virus carriers (hepatitis B carriers),30patients with chronic hepatitis B(chronic hepatitis B)，of which6cases are the mild CHB,9cases are the moderate CHB,15cases are the severe CHB,25cases of hepatitis B-related acute on chronic liver failureearly (HBV-acute on chronic liver failure early) and10healthy control (healthy control).Peripheral blood lymphocytes able to express perforin were analyzed byimmunohistochemistry. The expression of perforin mRNA in peripheral bloodmononuclear cells (PBMCs) was measured by SYBR GreenⅠ real-time quantitative PCR.The frequency of NK、NKT、HBV-specific CTL and the expression of perforin proteinwere measured by flow cytometry. Real-time florescence quantitative polymerase chainreaction (PCR) was used to detect serum HBV-DNA level. Serum ALT levels was detectedby the Backman CX-V automatic biochemical detector.Results:1. In the healthy control group, hepatitis B carriers, chronic hepatitis B andacute on chronic liver failure;1) the proportion of peripheral blood NK cells wererespectively (21.47±13.2)%、(20.19±8.08)%、(11.87±6.15)%and (5.18±3.69)%，SACLFand CHB patients were significantly lower than hepatitis B carriers and health control, thedifferent was statistically significant;2) the proportion of NKT wererespectively(18.16±9.33)%、(20.79±3.50)%、(14.28±6.74)%and(8.48±4.14)%, SACLFand CHB patients were significantly lower than hepatitis B carriers and health control, thedifferent was statistically significant;3) the proportion of non-specific CTL wererespectively(18.5±1.85)%、(17.72±5.89)%、(23.96±5.39)%and(28.83±5.02)%，SACLFand CHB patients were significantly higher than hepatitis B carriers and healthcontrol,the different was statistically significant;4) the proportion of specific CTL wererespectively(0.056±0.038)%、(0.065±0.053)%、(0.75±0.36)%and(0.35±0.18)%， CHBpatients were higher than hepatitis B carriers and health control,the different wasstatistically significant; SACLF was no different from CHB.The relation of the frequency of NK、NKT、non-special CTL and special CTL withALT were as follows: NK、NKT、CTL、Specific-CTL and serum ALT into correlation coefficient were r1=0.656(P1<0.01)，r2=0.57(P2<0.01)，r3=0.637(P3<0.01);r4=0.322，(p4<0.05).3. The relation of the frequency of NK、NKT、non-special CTL and special CTL withthe viral load were as follows: according to HBV-DNA levels,which were divided into twogroups, one was high viral group (28cases）：HBV-DNA≥10E4IU/ML;the other was lowviral load group(42cases):HBV-DNA<10E4IU/ML;NK%were respectively(8.85±2.95)%and (21.33±9.73)%(p=0.046); NKT%were respectively (11.35±6.52)%and (22.38±7.67)%(p=0.042); Non-specific CTL were respectively:(16.69±0.32)%and(28.15±0.35)%(p=0.043);specific CTL were respectively (0.85±0.23)%and(1.21±0.185)%(p=0.038).4. Immunohistochemistry showed that the expression of brown granules were thecells which secret perforin.5. In Peripheral blood PBMC of PFP mRNA gene expression levels:SACLF(0.54±0.78) and CHB(0.17±0.20) p=0.035;CHB (0.17±0.20) and hepatitis B carriers(0.061±0.05) p=0.041; hepatitis B carriers (0.061±0.05) and the healthy control(0.028±0.018) p=0.031; In the SACLF group of the death group(1.13±1.12) and thesurvival group(0.14±0.19) p=0.037;low viral load group(1.13±0.83) and the high viralload group (0.09±0.82) p=0.046, the difference was significant statistically.6.By the perforin antibody binding NK、NKT、specific-CTL cells:The frequency ofnon-specific CTL release perforin, SACLF(55.01±10.56)%is higher than CHB(25.04±5.67)%; the difference was statistically significant；The frequency of specific CTL releaseperforin, CHB(18.96±6.98)%is higher than Hepatitis B carriers (4.2±2.6)%; thedifference was statistically significant；The frequency of NK and NKT release perforin,SACLF(84.89±12.4)%and(57.47±1.55)%is higher than healthy control group(18.54±16.25)%and (8.1±4.53)%; the difference was statistically significant；Conclusion:1.Compared with the healthy controls、hepatitis B carriers, the frequencyof NK、NKT cells was significant lower than CHB、SACLF patients，but its function ofperforin expression was significantly higher, suggesting that when the HBV liver wasinflammation, NK and NKT performance high activity, its activity was relationship with liver damage.2. The frequency of HBV-specific CTL、non-specific CTL and the expression ofperforin with CHB was significantly higher, which was positively correlated with serumALT, negatively correlated with HBV-DNA, correlated with SACLF prognosis, suggestingthat the number of cytotoxic T cells and the expression of perforin was correlated withviral clearance and liver damage, which can be used as indicators of prognosis in SACLFsurvival effective.