卡介苗免疫小鼠T淋巴细胞亚群表达穿孔素与抗结核作用的相关性

目的 探讨卡介苗免疫小鼠抗结核作用和T细胞亚群表达穿孔素的相关性.方法 120只清洁级KM雄性小鼠分为2个对照组(各20只)、2个卡介苗组(各20只)、攻毒组(20只)和结核病组(20只).2个卡介苗组与攻毒组小鼠腹部皮内接种卡介苗,接种3个月后攻毒组和结核病组小鼠接受MTB攻击,同时对照1组和卡介苗1组小鼠取血后处死.攻毒组和结核病组小鼠于接受MTB攻毒1个月后与对照2组和卡介苗2组小鼠取血后处死.观察所有小鼠的肺、肝和脾组织病理学改变,并经组织匀浆涂片查找抗酸杆菌和MTB培养.流式细胞仪检测分析血液标本中表达穿孔素的T细胞亚群计数及其占总淋巴细胞百分率.多组间免疫学指标的比较采用单因素方差分析,两组间比较采用t检验.结果 结核病组小鼠的肺、肝和脾组织均有结核病表现,1个月内死亡11只,其他3组小鼠均未发现结核病表现,也无死亡鼠.卡介苗组小鼠表达穿孔素的CD8+T细胞数[(5.9±0.9)×103]明显高于对照组[(4.8±0.8)×103],差异有统计学意义(F=42.24,P＜0.01);结核病组小鼠表达穿孔素的CD+百分率[(5.6±0.9)%]明显低于卡介苗组[(7.3±1.1)%],差异有统计学意义(F=35.51,P＜0.05);攻毒组小鼠获得卡介苗免疫后,在致病量MTB攻击下表达穿孔素的CD3+、CD8+和CD4+ CD8+ T细胞数[(20.1±5.5)×103、(8.7±0.4)×103和72±19]及其占总淋巴细胞百分率[(23.3±3.3)%、(10.7±1.6)%和(0.084±0.015)%]均明显高于对照组[(11.1 ±3.0)×103、(4.8±0.8)×103和30±7及(14.9±1.7)%、(6.7±0.9)%和(0.040±0.006)%]、卡介苗组[(13.0±3.2)×103、(5.9±0.9)× 103和36±5及(15.5±1.7)%、(7.3±1.1)%和(0.044±0.007)%]及结核病组[(12.6±1.6)×103、(5.0±0.1)×103和31±3及(14.0±1.7)%、(5.6±0.9)%和(0.035±0.005)%],CD4+/CD8+比值(0.54±0.17)明显低于对照组(0.76±0.22),差异均有统计学意义(F值为4.54～74.98,均P＜0.05或P＜0.01).结论 卡介苗免疫主要诱导表达穿孔素的CD8+ T细胞水平升高,在致病量MTB攻击下卡介苗免疫小鼠不发生结核病,可能与表达穿孔素的CD3+、CD8+和CD4+ CD8+ T细胞水平升高相关;穿孔素表达水平高低可能是判断宿主抗结核免疫力强弱的重要标志之一.

Correlation between the expression of perforin in T-lymphocyte subsets and antituberculosis in mice vaccinated with bacillus calmette-guerin

Objective To investigate the relationship between the expression of perforin in Tlymphocyte subsets and antituberculosis in mice vaccinated with bacillus calmette-guerin (BCG). Methods 120 KM male mice were divided into a Control group (group C, n =40, 20 each in group C1 or C2), a BCG vaccinated group ( group B, n = 40, 20 each in group B1 or B2), a Tuberculosis group ( TB group,n =20), and a BT group, in which the mice were attacked by Mycobacterium tuberculosis H37 Rv (MTB)after acquired immunity by vaccination with BCG ( n = 20). Initially, mice in group B1, B2 and BT were vaccinated with BCG synchronously. Three month later, mice in group BT and TB were attacked by MTB synchronously. Samples of blood, lang, liver and spleen of mice in group C1 and B1 were collected at the same time. One month later again, Samples of blood, lung, liver and spleen of mice in group C2, B2, BT and TB were collected at the same time. Samples of blood were assayed for T-lymphocyte subsets expressing perforin (PFN+). The positive numbers of PFN+ [CD3+, CD4+, CD8+, CD4+ CD8+ double positive ( CD4+ CD8+ )] T lymphocytes and their percentages were assayed by flow cytometer. Specimens of lung, liver and spleen were examined for pathology and bacteriology. Results All the mice in the TB group acquired tuberculosis and the mortality was 55% (11/20) within 1 month. There were no tuberculosis and no death in mice of C, B and BT groups during the observation period. The amount of PFN+ CD8+ T lymphocytes in B group [(5.9 ±0.9) × 103] was significantly higher than that in C group [(4.8 ±0.8) × 103] (F =42.24,P ＜0.01 ). The PFN+ CD8+ % in TB group [(5.6 ± 0.9)%] was significantly less than that in B group [(7.3 ± 1.1)%] ( F = 35.51, P ＜ 0.05). For Mice in the BT group, the amount of PFN+ ( CD3+, CD8+,CD4+ CD8+ ) T lymphocytes [(20.1 ±5.5) × 103, (8.7 ±0.4) × 103, 72 ± 19] and their CD3+%, CD8+%,CD4+ CD8+% [(23.3 ± 3.3 ) %, ( 10.7 ± 1.6) %, (0.084 ± 0.015)%] were all higher than those in the B group [(13.0 ±3.2) ×103, (5.9 ±0.9) × 103, 36 ±5, (15.5 ± 1.7)%, (7.3 ± 1.1)%, (0.044 ±0.007)%] or the C group [(11.1 ±3.0) ×103, (4.8 ±0.8) ×103, 30 ±7, (14.9 ±1.7)%, (6.7 ±0.9)%, (0.040 ±0.006)%] or the TB group [(12.6 ±1.6) ×103, (5.0 ±0.1) ×103, 31 ±3,(14.0±1.7)%, (5.6±0.9)%, (0.035±0.005)%] (F=14.23-74.98, P＜0.01 or P＜0.05),and the CD4+/CD8+ in BT group (0.54 ±0.17) was significantly lower than that in C group (0.76 ±0.22)(F =4.54, P ＜ 0.01 ). Conclusions Pre-vaccination with BCG increased PFN+ CD8+ T lymphocytes in the host. Acquired immunity by BCG vaccination can protect the host from attack by Mycobacterium tuberculosis. Increased PFN+ (CD3+, CD8+ , CD4+ CD8+ ) T lymphocytes may be involved. The expression level of PFN by T lymphocytes may be an important marker for antituberculosis immunity.