CAJ | 학술논문

目的动态观察急性乙型肝炎(AHB)患者外周血HBcAg18-27表位特异性细胞毒性T淋巴细胞(CTL)、血清ALT、HBV DNA、HBsAg和淋巴细胞亚群的变化,探讨HBV特异性CTL频率的消长在病毒清除以及肝脏损伤中的作用.方法分别选取AHB、慢性乙型肝炎(CHB)患者外周血,根据人类白细胞抗原(HLA)-A0201结果分为两组:HLA-A0201阳性患者作为HBV特异性CTL检测组、HLA-A0201阴性患者作为特异性抗原表位对照组.用HLA-A0201限制性表位HBcAg18-27五聚体复合物通过流式细胞技术,动态定量检测外周血中HBV特异性CTL频率和T、B淋巴细胞与自然杀伤细胞(NK)和NKT淋巴细胞;以速率法检测血清ALT水平;荧光定量PCR检测HBV DNA水平; Abbott微粒子化学发光技术检测HBV血清学标志物.计量资料采用均数±标准差((x)±s)表示或中位数(P25-P75)描述,组间比较采用方差分析或非参数检验(KruskalWallis检验和Mann-Whitney U检验);两种计量指标的关系采用Pearson相关分析.结果 AHB患者入院第1、2、3周外周血HBcAg表位特异性CTL频率分别为2.11%(0.20%～3.64%)、3.56%(1.05%～5.91%)、2.03%(0.33%～3.58%),高于入院第4、5、6周的0.99%(0.12%～2.16%)、0.29%(0.05%～0.76%)、0.39%(0.05%～0.46%),也显著高于CHB的0.11%(0.06%～0.29%),z值分别为-3.258,-4.041,-3.259,P值均＜0.01.AHB患者HBcAg表位特异性CTL峰值延迟于血清HBV DNA、HBsAg和ALT等指标的峰值;在AHB患者中,HBcAg表位特异性CTL高频率患者的血清HBsAg消失时间早于CTL频率较低的患者[(1.75±1.04)周与(4.33±3.51)周,t=-2.018,P＜0.05].CD3+CD8+T淋巴细胞频率的峰值出现在入院后第2周,并与HBcAg表位特异性CTL峰值相重叠,两者动态变化规律呈相关性(r=0.420,P＜0.01).AHB患者早期外周血NK、NKT淋巴细胞数量显著低于正常对照组和CHB患者,但随着病情好转而逐渐恢复,AHB患者外周血NK细胞数量变化与HBcAg特异性CTL动态变化呈负相关(r=-0.435,P＜0.01).结论急性HBV感染者高频率的HBcAg表位特异性CTL与HBsAg的更早消失有密切关系,动态监测外周血中HBcAg特异性CTL频率变化,可以作为预测HBV感染后临床转归的参考指标;AHB患者外周血CD8+T淋巴细胞数量的变化,可以间接反应AHB患者体内HBcAg特异性CTL频率的改变. 目的動態觀察急性乙型肝炎(AHB)患者外週血HBcAg18-27錶位特異性細胞毒性T淋巴細胞(CTL)、血清ALT、HBV DNA、HBsAg和淋巴細胞亞群的變化,探討HBV特異性CTL頻率的消長在病毒清除以及肝髒損傷中的作用.方法分彆選取AHB、慢性乙型肝炎(CHB)患者外週血,根據人類白細胞抗原(HLA)-A0201結果分為兩組:HLA-A0201暘性患者作為HBV特異性CTL檢測組、HLA-A0201陰性患者作為特異性抗原錶位對照組.用HLA-A0201限製性錶位HBcAg18-27五聚體複閤物通過流式細胞技術,動態定量檢測外週血中HBV特異性CTL頻率和T、B淋巴細胞與自然殺傷細胞(NK)和NKT淋巴細胞;以速率法檢測血清ALT水平;熒光定量PCR檢測HBV DNA水平; Abbott微粒子化學髮光技術檢測HBV血清學標誌物.計量資料採用均數±標準差((x)±s)錶示或中位數(P25-P75)描述,組間比較採用方差分析或非參數檢驗(KruskalWallis檢驗和Mann-Whitney U檢驗);兩種計量指標的關繫採用Pearson相關分析.結果 AHB患者入院第1、2、3週外週血HBcAg錶位特異性CTL頻率分彆為2.11%(0.20%～3.64%)、3.56%(1.05%～5.91%)、2.03%(0.33%～3.58%),高于入院第4、5、6週的0.99%(0.12%～2.16%)、0.29%(0.05%～0.76%)、0.39%(0.05%～0.46%),也顯著高于CHB的0.11%(0.06%～0.29%),z值分彆為-3.258,-4.041,-3.259,P值均＜0.01.AHB患者HBcAg錶位特異性CTL峰值延遲于血清HBV DNA、HBsAg和ALT等指標的峰值;在AHB患者中,HBcAg錶位特異性CTL高頻率患者的血清HBsAg消失時間早于CTL頻率較低的患者[(1.75±1.04)週與(4.33±3.51)週,t=-2.018,P＜0.05].CD3+CD8+T淋巴細胞頻率的峰值齣現在入院後第2週,併與HBcAg錶位特異性CTL峰值相重疊,兩者動態變化規律呈相關性(r=0.420,P＜0.01).AHB患者早期外週血NK、NKT淋巴細胞數量顯著低于正常對照組和CHB患者,但隨著病情好轉而逐漸恢複,AHB患者外週血NK細胞數量變化與HBcAg特異性CTL動態變化呈負相關(r=-0.435,P＜0.01).結論急性HBV感染者高頻率的HBcAg錶位特異性CTL與HBsAg的更早消失有密切關繫,動態鑑測外週血中HBcAg特異性CTL頻率變化,可以作為預測HBV感染後臨床轉歸的參攷指標;AHB患者外週血CD8+T淋巴細胞數量的變化,可以間接反應AHB患者體內HBcAg特異性CTL頻率的改變.
목적 동태관찰급성을형간염(AHB)환자외주혈HBcAg18-27표위특이성세포독성T림파세포(CTL)、혈청ALT、HBV DNA、HBsAg화림파세포아군적변화,탐토HBV특이성CTL빈솔적소장재병독청제이급간장손상중적작용.방법 분별선취AHB、만성을형간염(CHB)환자외주혈,근거인류백세포항원(HLA)-A0201결과분위량조:HLA-A0201양성환자작위HBV특이성CTL검측조、HLA-A0201음성환자작위특이성항원표위대조조.용HLA-A0201한제성표위HBcAg18-27오취체복합물통과류식세포기술,동태정량검측외주혈중HBV특이성CTL빈솔화T、B림파세포여자연살상세포(NK)화NKT림파세포;이속솔법검측혈청ALT수평;형광정량PCR검측HBV DNA수평; Abbott미입자화학발광기술검측HBV혈청학표지물.계량자료채용균수±표준차((x)±s)표시혹중위수(P25-P75)묘술,조간비교채용방차분석혹비삼수검험(KruskalWallis검험화Mann-Whitney U검험);량충계량지표적관계채용Pearson상관분석.결과 AHB환자입원제1、2、3주외주혈HBcAg표위특이성CTL빈솔분별위2.11%(0.20%～3.64%)、3.56%(1.05%～5.91%)、2.03%(0.33%～3.58%),고우입원제4、5、6주적0.99%(0.12%～2.16%)、0.29%(0.05%～0.76%)、0.39%(0.05%～0.46%),야현저고우CHB적0.11%(0.06%～0.29%),z치분별위-3.258,-4.041,-3.259,P치균＜0.01.AHB환자HBcAg표위특이성CTL봉치연지우혈청HBV DNA、HBsAg화ALT등지표적봉치;재AHB환자중,HBcAg표위특이성CTL고빈솔환자적혈청HBsAg소실시간조우CTL빈솔교저적환자[(1.75±1.04)주여(4.33±3.51)주,t=-2.018,P＜0.05].CD3+CD8+T림파세포빈솔적봉치출현재입원후제2주,병여HBcAg표위특이성CTL봉치상중첩,량자동태변화규률정상관성(r=0.420,P＜0.01).AHB환자조기외주혈NK、NKT림파세포수량현저저우정상대조조화CHB환자,단수착병정호전이축점회복,AHB환자외주혈NK세포수량변화여HBcAg특이성CTL동태변화정부상관(r=-0.435,P＜0.01).결론 급성HBV감염자고빈솔적HBcAg표위특이성CTL여HBsAg적경조소실유밀절관계,동태감측외주혈중HBcAg특이성CTL빈솔변화,가이작위예측HBV감염후림상전귀적삼고지표;AHB환자외주혈CD8+T림파세포수량적변화,가이간접반응AHB환자체내HBcAg특이성CTL빈솔적개변.
Objective This report aims to investigate the dynamical changes of HBcAg18-27 epitope specific cytotoxic T lymphocytes(CTL), alanine aminotransferase (ALT), HBV DNA and HBsAg in peripheral blood of acute hepatitis B patients, and to explore the roles of HBcAg18-27-specific CTLs in virus clearance and liver injury. Methods Acute hepatitis B (AHB) and chronic hepatitis B (CHB) patients were divided into two groups according to results of HLA-A0201. Patients with positive HLA-A0201 were classified into HBcAg-specific CTL group and those with negative HLA-A0201 were referred as control group.The specific CTLs were stained with HLA-A0201 limited HBcAg18-27 epitope MHC-Pentamer and the frequencies of CTLs, T, B, NK and NKT cells were detected by flow cytometry (FCM). The serum ALT, HBV DNA and HBsAg were examined using speed analysis, quantitative PCR and abbott chemiluminescent technology. Results The frequencies of HBcAg18-27-specific CTLs in AHB patients were higher in the early three weeks as compared to the late three weeks. The apex time of HBV-specific CTL frequencies lagged behind those of HBV DNA, HBsAg and ALT. The loss of HBsAg in patients with high frequencies of HBVspecific CTL was earlier than that in patients with low frequencies (t = 2.018, P ＜ 0.05). In the second week the peak frequencies of CD3+CD8+ cells overlapped with that of HBcAg18-27-specific CTLs and with a positive correlation between (r = 0.420, P ＜ 0.05). During the early stages of AHB, the frequencies of NK and NKT cells were found significantly lower than that of control group and CHB group and the levels were back to normal after recovery. Moreover, a negative correlation existed between the frequencies of NK cells and the dynamic changes of HBcAg18-27-specific CTLs (r = -0.435, P ＜ 0.01) in AHB group. The frequencies of HBcAg18-27-specific CTLs were significantly higher as compared to CHB group in the first three weeks (z = -3.258, -4.04, and -3.259, P ＜ 0.01). Conclusion The early loss of HBsAg was closely related to the high frequencies of HBcAg18-27 specific CTLs in AHB patients. HBcAg-specific CTL frequencies in peripheral blood could be used to predict clinical outcome after HBV infection. The frequencie of CD8+ T cells can reflect the changes of frequencies of HBcAg-specific CTL. during acute HBV infection.