川崎病患者外周血淋巴细胞凋亡减少的初探
作者:易岂建 李成荣 杨锡强 刘恩梅
单位:重庆医科大学儿童医院,重庆400014
关键词:川崎病;细胞凋亡;IL-6
中国免疫学杂志990215 中国图书分类号 R593.2
摘 要 目的:探讨川崎病(KD)的免疫发病机理。方法:对26例KD患者和20名正常儿童外周血单个核细胞(PBMC)经anti-CD3诱导体外培养不同时间的凋亡进行计数凋亡细胞百分率和片段DNA分析。结果:KD患者凋亡细胞百分率和片段DNA出现时间较正常对照降低(P<0.001)和延迟,PBMC体外培养产生IL-6水平较正常对照显著升高(P<0.001);加抗IL-6单抗培养或静脉注射免疫球蛋白(IVIG)治疗可显著降低IL-6水平,逆转凋亡细胞百分率的降低和DNA片段化延迟。结论:KD患者PBMC凋亡下调可能与本病IL-6水平异常增高有关。
, 百拇医药
Preliminary study on the decreased apoptosis of lymphocytes in acute kawasaki disease
YI Qi-Jian,LI Cheng-Rong, YANG Xi-Qiang et al.
Children's Hospital Chongqing University of Medical Sciences,Chongqing 400014
Abstract Objective:To further explore the pathogenesis of kawasaki disease (KD).Methods:Calculating percentage of apoptotic cells and assaying DNA fragmentation in peripheral blood mononuclear cell(PBMC)stimulated by anti-CD3 for 0,12,24,48,72 h in vitro from 30 patients with KD and 20 age-matched health children.Results:Apoptotic cell percentage and DNA fragmentation were markedly decreased(P<0.001) and delayed (compared with those in normal controls),which could reach the states of control group only by 72 h cultured.Remarkabely increased production of IL-6 in cultured supernatants of PBMC induced by phytohemagglutinin(PHA) from KD patients was found in comparison with those of PBMC from controls(P<0.001).Adding anti-IL-6 mAb to the cultures or using intravenous immunoglobulin(IVIG)in vivo led to significantly decreased production of IL-6(P<0.001),and reversed decreased apoptotic cell percentage and delayed DNA fragmentation to be compatible with normal controls.Conclusion:Over-produced IL-6 might be involved in down-regulation of peripheral blood lymphocyte apoptosis in KD.
, 百拇医药
Key words Kawasaki disease Apoptosis IL-6
细胞凋亡(Apoptosis)是细胞生理性死亡的主要形式[1]。机体细胞增殖和死亡过程达到动态平衡是维持其自身恒定的必要条件。细胞凋亡过度或不足都会引起细胞数量失衡,导致临床疾病[2]。已推测凋亡可能与系统性红斑狼疮(SLE)等自身免疫性疾病有关。川崎病(Kawasaki disease,KD)是一原因不明的急性血管炎,其发病可能为感染或其他因素触发的异常免疫反应。本文拟对KD患者外周血单个核细胞(PBMC)体外培养时的凋亡进行观察,并对有关因素进行探讨。
1 材料与方法
1.1 研究对象 收集26例住院KD患者,均符合日本MCLS研究会诊断标准[3],随机分为两组,阿司匹林(ASP)治疗组和IVIG治疗组(IVIG+ASP)。IVIG组16例,男11例,女5例,年龄8.0个月~6.5岁(平均2.8±1.3岁),常规ASP治疗组10例,男6例,女4例,年龄6.5个月~5.5岁(平均2.5±1.2岁),两组患者开始治疗时病程长短、病情轻重均无明显差异。健康对照组20例,男12例,女8例,年龄1.0岁~6.2岁(平均2.6±1.2岁)。
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1.2 治疗方法 患者住院时病程为5.0~7.0 d,入院时立即给予治疗。IVIG组于平均病程5.8 d,使用IVIG治疗(2 g/kg,长春生物制品研究所,批号91-13),同时联合应用ASP 50 mg/(kg。d),退热后减至10 mg/(kg。d)。ASP组于平均病程6.2 d开始给予ASP治疗,剂量同前。
1.3 细胞分离及培养 两组患儿均在用药前和用药后3.0~5.0 d取肝素抗凝血,常规分离PBMC,调细胞浓度至1×106 ml-1,置24孔培养板,加anti-CD3(终浓度100 μg/ml),部分加抗IL-6单抗(第四军医大学免疫学教研室,终浓度1∶1 000),于37℃,5% CO2孵箱分别培养0、12、24、48、72 h。
1.4 凋亡细胞形态学观察 按照文献[4]方法,在荧光显微镜下观察,细胞体积缩小,核固缩,染色质凝固,沿核膜呈点状、新月形、杆状等定为凋亡细胞,计算200个细胞及凋亡细胞百分率。
, 百拇医药
1.5 DNA梯度观察 根据文献[5]方法略加改良,1×106 ml-1培养细胞中加入低渗性缓冲液(10 mmol/L Tris,1 mmol/l EDTA,0.2% TritonX-100,SIGMA公司)0.5 ml溶解细胞,离心取上清,用饱和酚(pH>7.8)及氯仿、异戊醇(24∶1)抽提,加无水乙醇,-20℃过夜。70%乙醇洗涤后,RNaseA(20 μg/ml,北京华美公司)去除RNA,1%琼脂糖凝胶电泳,紫外灯透照下观察片段DNA梯度,并照相记录。
1.6 IL-6的测定 无菌收集各种条件下PBMC经PHA(终浓度为20 μg/ml)刺激48 h后培养上清,采用双抗体夹心ELISA法检测。IL-6试剂盒为第四军医大学免疫学教研室产品。
2 结果
2.1 凋亡细胞百分率 正常对照组PBMC体外培养不同时间后可见细胞核固缩,呈新月形、杆状等凋亡典型的形态学改变,KD患者出现细胞形态学改变明显延迟。其中培养24、48 h后,KD患者PBMC凋亡百分率与正常对照组相比,结果见表1。
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经IVIG治疗后,KD患者PBMC体外培养凋亡细胞百分率明显提高,治疗前后差异显著;ASP组治疗前后无差别 ,两组治疗后比较有显著差异,见表2。
表1 KD与正常儿童PBMC的APO发生率比较(±s,%)
Tab.1 Percentage of apoptotic cell of PBMC from KD patients and normal subjects(±s,%) Groups
n
0 h
24 h
, 百拇医药
48 h
72 h
Normal
20
2.01±1.21
21.48±3.41
31.48±4.46
40.74±5.87
KD
30
2.26±1.071)
10.89±3.612)
, 百拇医药
18.67±5.68
33.15±8.45
Note:compared with normal 1)P>0.05,2)P<0.001表2 治疗前后KD患儿PBMC的APO发生率比较(±s,%)
Tab.2 Percentage of apoptotic cell of PBMC from KD patients before and after different treatment(±s,%) Groups
0 h
24 h
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48 h
72 h
Before
After
Before
After
Before
After
Before
After
IVIG+ASP
(n=16)
0
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0
12.57±3.721)
22.33±6.673)
20.00±4.121)
32.27±6.033)
34.57±7.321)
42.08±5.284)
ASP alone
(n=10)
0
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0
11.79±4.392)
13.48±3.98
19.28±4.482)
24.54±6.35
35.29±6.102)
38.58±5.47
Note:compared with after treatment 1)P<0.05,2)P>0.05;compared with after ASP alone treatment,3)P<0.05,4)P>0.05
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图1 不同条件DNA梯度
Fig.1 DNA ladder by 1% agarose gel electrophoresis in different conditions
Note:1.EcoRI/HindⅢ marker;2,3,4,5,6,7.PBMC from normal subject cultured for 0,12,24,48,72,96 h,respectively;8,9,10.PBMC from KD patient after IVIG cultured for 24,48,72 h,respectively;11.PBMC from KD cultured with anti-IL-6 for 48 h;16,15,14,13,12.PBMC from KD patient cultured for 0,12,24,48,72 h,respectively
, 百拇医药 当KD患者PBMC加入抗IL-6单抗培养,凋亡发生率(24、48 h 分别为20.83±7.98,29.33±8.19)显著增高,与未加抗IL-6单抗组比较(24、48 h 分别为11.52±6.86,19.10±6.07),差异显著(P<0.02,<0.01)。
2.2 片段DNA梯度 正常对照组PBMC经anti-CD3刺激培养24 h可见DNA片段化倾向,并随时间延长而明显,KD患者PBMC需培养72 h才有微量DNA片段。当IVIG治疗后及加入抗IL-6单抗培养时,DNA梯度出现时间明显提前,见图1。
2.3 IL-6水平测定 KD患者PBMC体外培养产生IL-6水平较正常对照组明显增高(t=6.885,P<0.001),当IVIG治疗后,IL-6水平降低,治疗前后差异显著(t=5.587,P<0.001);ASP治疗组治疗前后IL-6水平无差别(t=1.515,P<0.05);KD患者PBMC加入抗IL-6单抗培养,其上清IL-6水平显著降低,与未加抗IL-6单抗组比较,差异显著(t=6.334,P<0.001,见图2)。
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图2 不同组别IL-6水平
Fig.2 IL-6 level in PBMC supernatant of various groups
3 讨论
细胞凋亡与细胞的增殖、分化一样,属于最基本的细胞生物学行为,受到细胞内外多种因素的调节,在机体的发育与自身稳定中起重要作用[6]。凋亡异常与多种疾病密切相关,凋亡过多可引起免疫缺陷病和艾滋病,神经退行性变等,凋亡过少则与肿瘤等有关。近年提出凋亡被异常抑制可能与某些自身免疫性疾病有关[7]。
本研究以正常儿童PBMC为对照,探讨KD患者PBMC体外培养是否存在细胞凋亡常。Wesselborg和Reed实验发现,PBMC培养时加入PHA、anti-CD3(OKT3)或anti-TCR(BMAO31)单抗时,48 h开始出现凋亡的细胞形态学和DNA电泳改变[8,9]。本实验正常对照组PBMC体外培养结果与此相符。而KD患者PBMC体外培养时凋亡明显延迟,72 h凋亡细胞百分率和DNA片段化才接近正常24 h水平。表明活化的细胞克隆持续存活,免疫耐受遭到破坏。本文结果提示KD的发病除与T细胞的异常增殖活化和B细胞多克隆活化有关外,可能还与外周血淋巴细胞凋亡延迟有关。
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本文及文献报道KD患者血清及PBMC体外培养IL-6水平异常增高。IL-6可抑制野生型p53表达和p53诱导的骨髓白血病细胞的凋亡[10],刘佳等报道IL-6抑制小鼠(B杂交瘤)7TD1 细胞凋亡[11]。本研究通过体内IVIG治疗及体外PBMC培养加入抗IL-6单抗时,IL-6水平明显降低,凋亡细胞百分率增高及片段DNA梯度出现时间提前,提示KD患者PBMC发生凋亡降低或延迟可能与本病异常升高的IL-6有关。其机理尚待进一步研究。
作者简介:易岂建,男,31岁,博士,主治医师,主要从事小儿心脏免疫研究;
李成荣,男,45岁,教授,硕士生导师,主要从事分子生物学和肾脏免疫学研究
4 参考文献
1 Kerr J F, Wyllie A H,Carrie A R. Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics.Br J Cancer,1972;26:239
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2 Thompson C B.Apoptosis in the pathogenesis and treatment of disease.Science,1995;267:1456
3 川崎高作.新じ诊断の手引ぎ の解说.小儿科,1985;123:985
4 Mishell B B,Shiigi S M,Henry C et al. Preparation of mouse cell suspension.In:Mishell B B, Shiigi S M eds.Selected methods in cellular immunology.New York:W.H.Freeman,1980:21
5 Nigtorati G,Nicoletti L,Crocicchio F et al. Heat shock induces apoptosis in mouse thymocytes and protects them from glucocorticoid-induced cell death.Cellular Immunol,1992;143:348
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6 Steller H.Mechanisms and genes of cellular suicide.Science,1995;167:1445
7 Wu J G,Zhou T,Zhang J J et al. Correction of accelerated autoimmune disease by early replacement of the mutated lpr gene with the normal fas apoptosis gene in the T cells of transgenic MRL-lpr/lpr mice.Proc Natl Acad Sci USA,1994;91:2344
8 Wesselborg S,Janssen D, Kabelitz D. Induction of activation-driven death(apoptosis)in activated but not resting peripheral blood T cells.J Immunol,1993;150:4338
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9 Reed J C,Migashita T,Cuddy M et al. Regulation of p26-bcl-2 protein levels in human peripheral lymphocytes.Lab Inves,1992;67:443
10 Yonish-Rouach E,Resnitzky D,Lotern J et al. Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6.Nature,1991;352:345
11 刘 佳,李 宏,Hamou M F et al. IL-6对小鼠(B杂交瘤)7TD1细胞凋亡的调控作用.中华微生物学和免疫学杂志,1995;15(6):382
〔收稿1997-02-07 修回1997-12-29〕, 百拇医药
单位:重庆医科大学儿童医院,重庆400014
关键词:川崎病;细胞凋亡;IL-6
中国免疫学杂志990215 中国图书分类号 R593.2
摘 要 目的:探讨川崎病(KD)的免疫发病机理。方法:对26例KD患者和20名正常儿童外周血单个核细胞(PBMC)经anti-CD3诱导体外培养不同时间的凋亡进行计数凋亡细胞百分率和片段DNA分析。结果:KD患者凋亡细胞百分率和片段DNA出现时间较正常对照降低(P<0.001)和延迟,PBMC体外培养产生IL-6水平较正常对照显著升高(P<0.001);加抗IL-6单抗培养或静脉注射免疫球蛋白(IVIG)治疗可显著降低IL-6水平,逆转凋亡细胞百分率的降低和DNA片段化延迟。结论:KD患者PBMC凋亡下调可能与本病IL-6水平异常增高有关。
, 百拇医药
Preliminary study on the decreased apoptosis of lymphocytes in acute kawasaki disease
YI Qi-Jian,LI Cheng-Rong, YANG Xi-Qiang et al.
Children's Hospital Chongqing University of Medical Sciences,Chongqing 400014
Abstract Objective:To further explore the pathogenesis of kawasaki disease (KD).Methods:Calculating percentage of apoptotic cells and assaying DNA fragmentation in peripheral blood mononuclear cell(PBMC)stimulated by anti-CD3 for 0,12,24,48,72 h in vitro from 30 patients with KD and 20 age-matched health children.Results:Apoptotic cell percentage and DNA fragmentation were markedly decreased(P<0.001) and delayed (compared with those in normal controls),which could reach the states of control group only by 72 h cultured.Remarkabely increased production of IL-6 in cultured supernatants of PBMC induced by phytohemagglutinin(PHA) from KD patients was found in comparison with those of PBMC from controls(P<0.001).Adding anti-IL-6 mAb to the cultures or using intravenous immunoglobulin(IVIG)in vivo led to significantly decreased production of IL-6(P<0.001),and reversed decreased apoptotic cell percentage and delayed DNA fragmentation to be compatible with normal controls.Conclusion:Over-produced IL-6 might be involved in down-regulation of peripheral blood lymphocyte apoptosis in KD.
, 百拇医药
Key words Kawasaki disease Apoptosis IL-6
细胞凋亡(Apoptosis)是细胞生理性死亡的主要形式[1]。机体细胞增殖和死亡过程达到动态平衡是维持其自身恒定的必要条件。细胞凋亡过度或不足都会引起细胞数量失衡,导致临床疾病[2]。已推测凋亡可能与系统性红斑狼疮(SLE)等自身免疫性疾病有关。川崎病(Kawasaki disease,KD)是一原因不明的急性血管炎,其发病可能为感染或其他因素触发的异常免疫反应。本文拟对KD患者外周血单个核细胞(PBMC)体外培养时的凋亡进行观察,并对有关因素进行探讨。
1 材料与方法
1.1 研究对象 收集26例住院KD患者,均符合日本MCLS研究会诊断标准[3],随机分为两组,阿司匹林(ASP)治疗组和IVIG治疗组(IVIG+ASP)。IVIG组16例,男11例,女5例,年龄8.0个月~6.5岁(平均2.8±1.3岁),常规ASP治疗组10例,男6例,女4例,年龄6.5个月~5.5岁(平均2.5±1.2岁),两组患者开始治疗时病程长短、病情轻重均无明显差异。健康对照组20例,男12例,女8例,年龄1.0岁~6.2岁(平均2.6±1.2岁)。
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1.2 治疗方法 患者住院时病程为5.0~7.0 d,入院时立即给予治疗。IVIG组于平均病程5.8 d,使用IVIG治疗(2 g/kg,长春生物制品研究所,批号91-13),同时联合应用ASP 50 mg/(kg。d),退热后减至10 mg/(kg。d)。ASP组于平均病程6.2 d开始给予ASP治疗,剂量同前。
1.3 细胞分离及培养 两组患儿均在用药前和用药后3.0~5.0 d取肝素抗凝血,常规分离PBMC,调细胞浓度至1×106 ml-1,置24孔培养板,加anti-CD3(终浓度100 μg/ml),部分加抗IL-6单抗(第四军医大学免疫学教研室,终浓度1∶1 000),于37℃,5% CO2孵箱分别培养0、12、24、48、72 h。
1.4 凋亡细胞形态学观察 按照文献[4]方法,在荧光显微镜下观察,细胞体积缩小,核固缩,染色质凝固,沿核膜呈点状、新月形、杆状等定为凋亡细胞,计算200个细胞及凋亡细胞百分率。
, 百拇医药
1.5 DNA梯度观察 根据文献[5]方法略加改良,1×106 ml-1培养细胞中加入低渗性缓冲液(10 mmol/L Tris,1 mmol/l EDTA,0.2% TritonX-100,SIGMA公司)0.5 ml溶解细胞,离心取上清,用饱和酚(pH>7.8)及氯仿、异戊醇(24∶1)抽提,加无水乙醇,-20℃过夜。70%乙醇洗涤后,RNaseA(20 μg/ml,北京华美公司)去除RNA,1%琼脂糖凝胶电泳,紫外灯透照下观察片段DNA梯度,并照相记录。
1.6 IL-6的测定 无菌收集各种条件下PBMC经PHA(终浓度为20 μg/ml)刺激48 h后培养上清,采用双抗体夹心ELISA法检测。IL-6试剂盒为第四军医大学免疫学教研室产品。
2 结果
2.1 凋亡细胞百分率 正常对照组PBMC体外培养不同时间后可见细胞核固缩,呈新月形、杆状等凋亡典型的形态学改变,KD患者出现细胞形态学改变明显延迟。其中培养24、48 h后,KD患者PBMC凋亡百分率与正常对照组相比,结果见表1。
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经IVIG治疗后,KD患者PBMC体外培养凋亡细胞百分率明显提高,治疗前后差异显著;ASP组治疗前后无差别 ,两组治疗后比较有显著差异,见表2。
表1 KD与正常儿童PBMC的APO发生率比较(±s,%)
Tab.1 Percentage of apoptotic cell of PBMC from KD patients and normal subjects(±s,%) Groups
n
0 h
24 h
, 百拇医药
48 h
72 h
Normal
20
2.01±1.21
21.48±3.41
31.48±4.46
40.74±5.87
KD
30
2.26±1.071)
10.89±3.612)
, 百拇医药
18.67±5.68
33.15±8.45
Note:compared with normal 1)P>0.05,2)P<0.001表2 治疗前后KD患儿PBMC的APO发生率比较(±s,%)
Tab.2 Percentage of apoptotic cell of PBMC from KD patients before and after different treatment(±s,%) Groups
0 h
24 h
, 百拇医药
48 h
72 h
Before
After
Before
After
Before
After
Before
After
IVIG+ASP
(n=16)
0
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0
12.57±3.721)
22.33±6.673)
20.00±4.121)
32.27±6.033)
34.57±7.321)
42.08±5.284)
ASP alone
(n=10)
0
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0
11.79±4.392)
13.48±3.98
19.28±4.482)
24.54±6.35
35.29±6.102)
38.58±5.47
Note:compared with after treatment 1)P<0.05,2)P>0.05;compared with after ASP alone treatment,3)P<0.05,4)P>0.05
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图1 不同条件DNA梯度
Fig.1 DNA ladder by 1% agarose gel electrophoresis in different conditions
Note:1.EcoRI/HindⅢ marker;2,3,4,5,6,7.PBMC from normal subject cultured for 0,12,24,48,72,96 h,respectively;8,9,10.PBMC from KD patient after IVIG cultured for 24,48,72 h,respectively;11.PBMC from KD cultured with anti-IL-6 for 48 h;16,15,14,13,12.PBMC from KD patient cultured for 0,12,24,48,72 h,respectively
, 百拇医药 当KD患者PBMC加入抗IL-6单抗培养,凋亡发生率(24、48 h 分别为20.83±7.98,29.33±8.19)显著增高,与未加抗IL-6单抗组比较(24、48 h 分别为11.52±6.86,19.10±6.07),差异显著(P<0.02,<0.01)。
2.2 片段DNA梯度 正常对照组PBMC经anti-CD3刺激培养24 h可见DNA片段化倾向,并随时间延长而明显,KD患者PBMC需培养72 h才有微量DNA片段。当IVIG治疗后及加入抗IL-6单抗培养时,DNA梯度出现时间明显提前,见图1。
2.3 IL-6水平测定 KD患者PBMC体外培养产生IL-6水平较正常对照组明显增高(t=6.885,P<0.001),当IVIG治疗后,IL-6水平降低,治疗前后差异显著(t=5.587,P<0.001);ASP治疗组治疗前后IL-6水平无差别(t=1.515,P<0.05);KD患者PBMC加入抗IL-6单抗培养,其上清IL-6水平显著降低,与未加抗IL-6单抗组比较,差异显著(t=6.334,P<0.001,见图2)。
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图2 不同组别IL-6水平
Fig.2 IL-6 level in PBMC supernatant of various groups
3 讨论
细胞凋亡与细胞的增殖、分化一样,属于最基本的细胞生物学行为,受到细胞内外多种因素的调节,在机体的发育与自身稳定中起重要作用[6]。凋亡异常与多种疾病密切相关,凋亡过多可引起免疫缺陷病和艾滋病,神经退行性变等,凋亡过少则与肿瘤等有关。近年提出凋亡被异常抑制可能与某些自身免疫性疾病有关[7]。
本研究以正常儿童PBMC为对照,探讨KD患者PBMC体外培养是否存在细胞凋亡常。Wesselborg和Reed实验发现,PBMC培养时加入PHA、anti-CD3(OKT3)或anti-TCR(BMAO31)单抗时,48 h开始出现凋亡的细胞形态学和DNA电泳改变[8,9]。本实验正常对照组PBMC体外培养结果与此相符。而KD患者PBMC体外培养时凋亡明显延迟,72 h凋亡细胞百分率和DNA片段化才接近正常24 h水平。表明活化的细胞克隆持续存活,免疫耐受遭到破坏。本文结果提示KD的发病除与T细胞的异常增殖活化和B细胞多克隆活化有关外,可能还与外周血淋巴细胞凋亡延迟有关。
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本文及文献报道KD患者血清及PBMC体外培养IL-6水平异常增高。IL-6可抑制野生型p53表达和p53诱导的骨髓白血病细胞的凋亡[10],刘佳等报道IL-6抑制小鼠(B杂交瘤)7TD1 细胞凋亡[11]。本研究通过体内IVIG治疗及体外PBMC培养加入抗IL-6单抗时,IL-6水平明显降低,凋亡细胞百分率增高及片段DNA梯度出现时间提前,提示KD患者PBMC发生凋亡降低或延迟可能与本病异常升高的IL-6有关。其机理尚待进一步研究。
作者简介:易岂建,男,31岁,博士,主治医师,主要从事小儿心脏免疫研究;
李成荣,男,45岁,教授,硕士生导师,主要从事分子生物学和肾脏免疫学研究
4 参考文献
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9 Reed J C,Migashita T,Cuddy M et al. Regulation of p26-bcl-2 protein levels in human peripheral lymphocytes.Lab Inves,1992;67:443
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〔收稿1997-02-07 修回1997-12-29〕, 百拇医药