一个新的T细胞活化分子（CD226）与HIV感染/艾滋病的相关性研究
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摘要
实验目的
1．明确HIV／AIDS患者T淋巴细胞以及自然杀伤(NK)细胞膜表面CD226(mCD226)分子表达水平，与其它免疫活化指标水平变化相比较的特点；
2．明确HIV／AIDS患者血浆中可溶性CD226(sCD226)分子水平与mCD226表达变化的相关性；
3．明确mCD226／sCD226分子水平在评价HIV感染者机体免疫状态和疾病进程中的临床意义。
实验材料与方法
一、实验材料
1．材料：
LeoAl(CD226-FITC)单克隆抗体(mAb)由第四军医大学免疫学教研室提供，抗CD3、CD4、CD8、CD16、HLA-DR的mAb、CD4 T淋巴细胞绝对计数试剂盒(TruCount~(TM))以及流式细胞仪(FACSCalibur)均为美国Becton Dick-inson公司产品。HIV-1／2抗体和HIV-1 p24抗原检测试剂均为荷兰Orga-non Teknika公司产品，免疫印迹(WB)试验试剂为新加坡Genelabs Diagnos-tics公司产品。RPMI 1640为GIBCO公司产品，PHA和rIL-2为Sigma公司产品，其它化学试剂均为国产分析纯。
2．研究对象：
34例HIV-1抗体阳性病例均经中国医科大学第一临床学院艾滋病研究室WB试验确认，其中男性26例，女性8例，年龄范围在18-40岁，根据1993年美国CDC制定的艾滋病监测病例分类及扩大的诊断标准，无症状
HIV感染(AS)组(CD4+T淋巴细胞妻200个/闪)25例，AIDS组(CD4+T
淋巴细胞<200个/川)9例。感染途径包括输血、静脉注毒和性传播。所
有外周静脉血均在未接受抗逆转录病毒治疗前采集并保存，EDTA一KZ抗凝。
正常对照组为随机抽取的26例健康人，男性16例，女性ro例，年龄范围在
1942岁。
二、实验方法
1.cD4+T淋巴细胞绝对计数:
参照美国CDc“HIV感染者cD4+T细胞的检测指导方法〔‘，〕”，采用溶
血一免洗法TruCOUNTTM进行FITC/PE/Pe尤P三色荧光标记染色:T加COU-
NTTM测试管中，加20闪TriTEsT试剂(CD4/CDS/CD3诚b)和50闪抗凝
血，孵育巧而n后再加450闪免洗溶血素，孵育巧一30而n。流式细胞术
(FCM)检测和分析:应用M血isET软件进行，得到CD3+T细胞、CD4+T
细胞、CDS十T细胞绝对数以及相对比值。
2.淋巴细胞膜表面CD226(mCD226)分子的检测:
采用三荧光染色标记体外培养细胞、HIV感染者以及健康对照的全血
细胞，按CD226/CDS/CD3，CD226/CD4/CD3，HLA一DR/CDS/CD3，HLA-
DR/CD4/CD3，CD226/CD16/CD3、CD226/CDS/CD3和CD226/CD咚/CD3等
mAb组合给试管编号并在管中分别加人一定量的上述mAb;再加人全血
80闪或细胞悬液，室温保存30而n;加2一而溶血素，室温保存5一10min至完
全溶血;重复洗细胞3次后，加0.5而1%多聚甲醛固定细胞，避光保存，
FCM检测与分析。应用CellQuest软件。用前向散射角(FSC)及侧向散射
角(SSC)确定淋巴细胞群，再分别以CD3+细胞、CD3·CD16+、CD3一CDS+
淋巴细胞为门，分析表达CD226的细胞，以阳性百分率(%)表示，并根据淋
巴细胞数计算出特定细胞亚群绝对数(。ells/~，)。
3.双抗体夹心EUSA法检测可溶性CD226(sCD226)分子水平:
玩。Al包被EllSA板，过夜。向板中加人标准品CD226/F。融合蛋白
和待测样本，孵育45而n，再加人HRP标记的mAb，孵育45min，加人底物
TMB显色，2.smoFL HZSO4终止反应。测定OD4s。吸光度(A)，以标准品的
A值和相应的浓度作标准曲线;将待测样本的A值与标准曲线相比，计算
待测样本的sCD226浓度。
4.HIV一1 503体外活化外周血单个核细胞(PBMC):
Hlv一1。。3株和MT4细胞系由中国疾病预防与控制中心参比实验室惠
赠。按常规传代并测定感染滴度(TCID50)。分离健康人PBMC，调整细胞
浓度后分组用于实验，pBMC与HIV一1 503(125 TCID，)共孵育Zh后，接种
于细胞培养板，作为染毒组，设立不染毒或不加PHA的对照组。在不同时
间点收集培养细胞进行免疫荧光染色，FCM分析，收集细胞培养上清检测
SCD226的浓度，同时测定HIV一1 p24抗原，以确定细胞的病毒感染效果。
5.统计学分析:
用SPSS 10.0软件对实验数据进行分析。t检验进行As/AIDS患者和
健康对照组间sCD226、mCD226均值比较。Spe一相关分析sCD226分
子水平与CD226+T细胞、CD4+T细胞百分率的相关性。
实验结果
一、HIWAIDS患者T淋巴细胞CD226的表达水平
经FCM测定外周血T淋巴细胞及其CD4+T细胞、CDS+T细胞亚群
中的CD226抗原表达可见:AS组和AIDS组CD3+T细胞中CD226+细胞
百分率为41 .6土31 .4%和47.3土17.2%，明显高于健康对照组22.6*16.
7%(p<0 .01);CD4+T细胞和CDS+T细胞亚群中CD226+细胞百分率
均显著高于健康对照(p<0.01)，但与CD3+T细胞中的CD226表达水平
没有显著差异;CD4+T细胞与CDS+T细胞亚群，AS组与AIDS组CD226
+细胞百分率差异均不显著。
二、HIWAIDS患者T淋巴细胞CD226和HLA一DR的表达
CD226+T淋巴细胞百分率在AS组(41 .6士31 .4%)和AIDS组(47.3
士17.2%)均显著高于健康对照组(22.6士16.7%)，HLA一DR+T淋巴细胞
百分率只在AIDS组(44.2土24.1%)与健康对照组(14.1土5.5%)差异显
著，AS组(19.7土6.8%)与健康对照无显著差异。
三、人PBMc CD226和HLA一D
Objective
1. To investigate the level of CD226 expression on T lymphocytes (mCD226 ) and NK cells from HIV/AIDS individuals, and its characteristics in comparing with other immune activation markers.
2. To demonstrate the correlation between soluble CD226 ( sCD226) and mCD226 expression in HIV /AIDS individuals.
3. To make sure the clinical significants of mCD226/sCD226 in evaluating immune status and disease progression of HIV /AIDS patients.
Materials and Methods
1. Reagents and instruments
Fluorescein isothiocyanate (FITC) conjugated monoclonal antibody (mAb) against GD226 ( LeoAl) was kindly provided by prof. Jin from Fourth Military Medical University. Phycoerythrin ( PE) and Peridinin chlorophylla protein (PerCP) conjugated anti- CD3, CD4, CD8, CD16 mAb , CD4 T lymphocyte absolute count kit ( TruCount ) and flow cytometry ( FACSCalibur) were all products from Becton Dickinson immuno-cytometry system ( USA). The kits used to detect HIV-1/2 antibodies and HFV-1 antigen (p24) was purchased from Or-ganon Teknika Company ( Holland) , Western Blot ( WB ) test reagents were products of Genelabs Diagnostics Company (Singapore). RPMI-1640 was from GIBCO Company, PHA and rIL-2 were from Sigma Company.
2. Study Subjects
34 of HIV-1 positive individuals included in the study were screened out by
using the antibody detection kits, and confirmed through WB test in our AIDS research center. 26 males and 8 females, range from 18 to 40 years old, Ac-ccording to the United States center for disease control (CDC) criteria for HIV infection disease (1993 ) , HIV seropositive individuals should be stratified a-symptomatic stage ( AS) ( CD4+ T lymphocyte greater than 200/ul) and AIDS stage (CD4+T less than 200/ul) , 25 cases were in AS, 9 cases in AIDS stage , they were infected through blood transfusion, drug abuse and sex course,no receiving anti-retroviral therapy before the studying. 26 healthy adult volunteers were selected as the control, including 16 males and 10 females, age is from 18 to 42 years old. Peripheral venous blood was drawn from HIV infected /AIDS patients and healthy control, and put into EDTA-K2 containing tubes.
3. Peripheral blood mononuclear cells ( PBMC) infected by HIV-1 strain in vitro
HIV-Isr33 strain and MT-4 cell line ware kind gifts from Reference Laboratory in CDC China, The infectious liter (TCID^ ) was examined according to routine procedure. PBMC were separated from healthy individuals, cells (106yjV ml ) were divided into several groups for further test, one group was mixed with HIV-1SF33( 125 TCIDso) culture for2h, wash twice, such cells were cultured, at the mean time establish control groups with or without HIV-1 and /or PHA mixed. Determined HIV-1 p24 antigen by collecting cultural supernatant to make sure the infectious effect on PBMCs.
4. Immunofluorescence Stain and analysis by flow cytometry (FCM)
To perform CD4 + T lymphocyte absolute counts; using a no-wash procedure with TruCOUNT kit, analysis by MultiSET software. To analysis NK related phenotypes by using a lyse procedure: different fluorochrome-conjugated mAbs were combinated as following: CD226/CD16/CD3, CD226/CD8/CD3, CD226/CD4/CD3. Adding 6ul of each mAb into Falcon tubes as the combination shown above, 80ul whole blood was added, after a 30min incubation at room temperature, treating the stained sample with Lysing Solution to lyse eryth-rocytes, then wash the sample twice with PBS to remove excess antibody and debris, resuspended in 0. 5ml phosphate buffered saline (PBS) solution consist of 0.5% polyformaldehyde, Finally, analyze the stained cells by FCM CellQuest
software, calculate the percentage ( % ) of antigen expression and specific subset absolute count ( cells/ mm3 ).
5. Double antibody sandwich ELISA for the detection of sCD226
Microtitre plates were coated with mAb-LeoAl, incubated at 4t for 24 h, wash plates two times. Add the test sample/standard CD226 Fc protein, add HRP-conjugated mAb, incubate at room temperature for 45 mins. Add freshly prepared
1．明确HIV／AIDS患者T淋巴细胞以及自然杀伤(NK)细胞膜表面CD226(mCD226)分子表达水平，与其它免疫活化指标水平变化相比较的特点；
2．明确HIV／AIDS患者血浆中可溶性CD226(sCD226)分子水平与mCD226表达变化的相关性；
3．明确mCD226／sCD226分子水平在评价HIV感染者机体免疫状态和疾病进程中的临床意义。
实验材料与方法
一、实验材料
1．材料：
LeoAl(CD226-FITC)单克隆抗体(mAb)由第四军医大学免疫学教研室提供，抗CD3、CD4、CD8、CD16、HLA-DR的mAb、CD4 T淋巴细胞绝对计数试剂盒(TruCount~(TM))以及流式细胞仪(FACSCalibur)均为美国Becton Dick-inson公司产品。HIV-1／2抗体和HIV-1 p24抗原检测试剂均为荷兰Orga-non Teknika公司产品，免疫印迹(WB)试验试剂为新加坡Genelabs Diagnos-tics公司产品。RPMI 1640为GIBCO公司产品，PHA和rIL-2为Sigma公司产品，其它化学试剂均为国产分析纯。
2．研究对象：
34例HIV-1抗体阳性病例均经中国医科大学第一临床学院艾滋病研究室WB试验确认，其中男性26例，女性8例，年龄范围在18-40岁，根据1993年美国CDC制定的艾滋病监测病例分类及扩大的诊断标准，无症状
HIV感染(AS)组(CD4+T淋巴细胞妻200个/闪)25例，AIDS组(CD4+T
淋巴细胞<200个/川)9例。感染途径包括输血、静脉注毒和性传播。所
有外周静脉血均在未接受抗逆转录病毒治疗前采集并保存，EDTA一KZ抗凝。
正常对照组为随机抽取的26例健康人，男性16例，女性ro例，年龄范围在
1942岁。
二、实验方法
1.cD4+T淋巴细胞绝对计数:
参照美国CDc“HIV感染者cD4+T细胞的检测指导方法〔‘，〕”，采用溶
血一免洗法TruCOUNTTM进行FITC/PE/Pe尤P三色荧光标记染色:T加COU-
NTTM测试管中，加20闪TriTEsT试剂(CD4/CDS/CD3诚b)和50闪抗凝
血，孵育巧而n后再加450闪免洗溶血素，孵育巧一30而n。流式细胞术
(FCM)检测和分析:应用M血isET软件进行，得到CD3+T细胞、CD4+T
细胞、CDS十T细胞绝对数以及相对比值。
2.淋巴细胞膜表面CD226(mCD226)分子的检测:
采用三荧光染色标记体外培养细胞、HIV感染者以及健康对照的全血
细胞，按CD226/CDS/CD3，CD226/CD4/CD3，HLA一DR/CDS/CD3，HLA-
DR/CD4/CD3，CD226/CD16/CD3、CD226/CDS/CD3和CD226/CD咚/CD3等
mAb组合给试管编号并在管中分别加人一定量的上述mAb;再加人全血
80闪或细胞悬液，室温保存30而n;加2一而溶血素，室温保存5一10min至完
全溶血;重复洗细胞3次后，加0.5而1%多聚甲醛固定细胞，避光保存，
FCM检测与分析。应用CellQuest软件。用前向散射角(FSC)及侧向散射
角(SSC)确定淋巴细胞群，再分别以CD3+细胞、CD3·CD16+、CD3一CDS+
淋巴细胞为门，分析表达CD226的细胞，以阳性百分率(%)表示，并根据淋
巴细胞数计算出特定细胞亚群绝对数(。ells/~，)。
3.双抗体夹心EUSA法检测可溶性CD226(sCD226)分子水平:
玩。Al包被EllSA板，过夜。向板中加人标准品CD226/F。融合蛋白
和待测样本，孵育45而n，再加人HRP标记的mAb，孵育45min，加人底物
TMB显色，2.smoFL HZSO4终止反应。测定OD4s。吸光度(A)，以标准品的
A值和相应的浓度作标准曲线;将待测样本的A值与标准曲线相比，计算
待测样本的sCD226浓度。
4.HIV一1 503体外活化外周血单个核细胞(PBMC):
Hlv一1。。3株和MT4细胞系由中国疾病预防与控制中心参比实验室惠
赠。按常规传代并测定感染滴度(TCID50)。分离健康人PBMC，调整细胞
浓度后分组用于实验，pBMC与HIV一1 503(125 TCID，)共孵育Zh后，接种
于细胞培养板，作为染毒组，设立不染毒或不加PHA的对照组。在不同时
间点收集培养细胞进行免疫荧光染色，FCM分析，收集细胞培养上清检测
SCD226的浓度，同时测定HIV一1 p24抗原，以确定细胞的病毒感染效果。
5.统计学分析:
用SPSS 10.0软件对实验数据进行分析。t检验进行As/AIDS患者和
健康对照组间sCD226、mCD226均值比较。Spe一相关分析sCD226分
子水平与CD226+T细胞、CD4+T细胞百分率的相关性。
实验结果
一、HIWAIDS患者T淋巴细胞CD226的表达水平
经FCM测定外周血T淋巴细胞及其CD4+T细胞、CDS+T细胞亚群
中的CD226抗原表达可见:AS组和AIDS组CD3+T细胞中CD226+细胞
百分率为41 .6土31 .4%和47.3土17.2%，明显高于健康对照组22.6*16.
7%(p<0 .01);CD4+T细胞和CDS+T细胞亚群中CD226+细胞百分率
均显著高于健康对照(p<0.01)，但与CD3+T细胞中的CD226表达水平
没有显著差异;CD4+T细胞与CDS+T细胞亚群，AS组与AIDS组CD226
+细胞百分率差异均不显著。
二、HIWAIDS患者T淋巴细胞CD226和HLA一DR的表达
CD226+T淋巴细胞百分率在AS组(41 .6士31 .4%)和AIDS组(47.3
士17.2%)均显著高于健康对照组(22.6士16.7%)，HLA一DR+T淋巴细胞
百分率只在AIDS组(44.2土24.1%)与健康对照组(14.1土5.5%)差异显
著，AS组(19.7土6.8%)与健康对照无显著差异。
三、人PBMc CD226和HLA一D
Objective
1. To investigate the level of CD226 expression on T lymphocytes (mCD226 ) and NK cells from HIV/AIDS individuals, and its characteristics in comparing with other immune activation markers.
2. To demonstrate the correlation between soluble CD226 ( sCD226) and mCD226 expression in HIV /AIDS individuals.
3. To make sure the clinical significants of mCD226/sCD226 in evaluating immune status and disease progression of HIV /AIDS patients.
Materials and Methods
1. Reagents and instruments
Fluorescein isothiocyanate (FITC) conjugated monoclonal antibody (mAb) against GD226 ( LeoAl) was kindly provided by prof. Jin from Fourth Military Medical University. Phycoerythrin ( PE) and Peridinin chlorophylla protein (PerCP) conjugated anti- CD3, CD4, CD8, CD16 mAb , CD4 T lymphocyte absolute count kit ( TruCount ) and flow cytometry ( FACSCalibur) were all products from Becton Dickinson immuno-cytometry system ( USA). The kits used to detect HIV-1/2 antibodies and HFV-1 antigen (p24) was purchased from Or-ganon Teknika Company ( Holland) , Western Blot ( WB ) test reagents were products of Genelabs Diagnostics Company (Singapore). RPMI-1640 was from GIBCO Company, PHA and rIL-2 were from Sigma Company.
2. Study Subjects
34 of HIV-1 positive individuals included in the study were screened out by
using the antibody detection kits, and confirmed through WB test in our AIDS research center. 26 males and 8 females, range from 18 to 40 years old, Ac-ccording to the United States center for disease control (CDC) criteria for HIV infection disease (1993 ) , HIV seropositive individuals should be stratified a-symptomatic stage ( AS) ( CD4+ T lymphocyte greater than 200/ul) and AIDS stage (CD4+T less than 200/ul) , 25 cases were in AS, 9 cases in AIDS stage , they were infected through blood transfusion, drug abuse and sex course,no receiving anti-retroviral therapy before the studying. 26 healthy adult volunteers were selected as the control, including 16 males and 10 females, age is from 18 to 42 years old. Peripheral venous blood was drawn from HIV infected /AIDS patients and healthy control, and put into EDTA-K2 containing tubes.
3. Peripheral blood mononuclear cells ( PBMC) infected by HIV-1 strain in vitro
HIV-Isr33 strain and MT-4 cell line ware kind gifts from Reference Laboratory in CDC China, The infectious liter (TCID^ ) was examined according to routine procedure. PBMC were separated from healthy individuals, cells (106yjV ml ) were divided into several groups for further test, one group was mixed with HIV-1SF33( 125 TCIDso) culture for2h, wash twice, such cells were cultured, at the mean time establish control groups with or without HIV-1 and /or PHA mixed. Determined HIV-1 p24 antigen by collecting cultural supernatant to make sure the infectious effect on PBMCs.
4. Immunofluorescence Stain and analysis by flow cytometry (FCM)
To perform CD4 + T lymphocyte absolute counts; using a no-wash procedure with TruCOUNT kit, analysis by MultiSET software. To analysis NK related phenotypes by using a lyse procedure: different fluorochrome-conjugated mAbs were combinated as following: CD226/CD16/CD3, CD226/CD8/CD3, CD226/CD4/CD3. Adding 6ul of each mAb into Falcon tubes as the combination shown above, 80ul whole blood was added, after a 30min incubation at room temperature, treating the stained sample with Lysing Solution to lyse eryth-rocytes, then wash the sample twice with PBS to remove excess antibody and debris, resuspended in 0. 5ml phosphate buffered saline (PBS) solution consist of 0.5% polyformaldehyde, Finally, analyze the stained cells by FCM CellQuest
software, calculate the percentage ( % ) of antigen expression and specific subset absolute count ( cells/ mm3 ).
5. Double antibody sandwich ELISA for the detection of sCD226
Microtitre plates were coated with mAb-LeoAl, incubated at 4t for 24 h, wash plates two times. Add the test sample/standard CD226 Fc protein, add HRP-conjugated mAb, incubate at room temperature for 45 mins. Add freshly prepared
引文
1. T cell activation is associated with lower CD4 + T cell gains in human immunodeficiency virus-infected patients with sustained viral suppression during antiretroviral therapy. J Infect Dis. 2003 May 15; 187(10): 1534-43.
2. Leng Q, Borkow G, Weisman Z, et al. Immune activation correlates better than HIV plasma viral load with CD4 T-cell decline during HIV infection. J Acquir Immune Defic Syndr. 2001 Aug 1; 27(4): 389-97.
3. Hazenberg MD, Otto SA, van Benthem BH, et al. Persistent immune activation in HIV-1 infection is associated with progression to AIDS. AIDS. 2003 Sep 5; 17(13): 1881-8.
4. Savarino A, Bottarel F, Malavasi F, et al. Role of CD38 in HIV-1 infection: an epiphenomenon of T-cell activation or an active player in virus/host interactions? AIDS,2000; 14 (9): 1079-89.
5. Jia W, Lin XS, Zhu H, et al. Preparation and characterization of mabs against different epitopes of CD226 (PTA1). Hybridoma, 2000, 19 (5): 489-494.
6．田方，金伯泉、姜绍淳，等．一种新的免疫球蛋白超家族成员PTAI的细胞分布及表达研究．细胞与分子免疫学杂志，1996；12：53-57．
7. Sherrington PD, Scott JL, Jin B, et al. TliSA1 (PTA1) activation antigen implicated in T cell differentiation and platelet activation is a member of th immunoglobulin super family exhibiting distinctive regulation of expression. J Biol Chem 1997,272:21735-21744.
8. Chen L, Xie X, Zhang X, et al. The expression, regulation and adhesion function of a novel CD molecule, CD226, on human endothelial cells, Life Sci. 2003 Sep 19; 73 (18): 2373-82.
9. Bottino C, Castriconi R, Pende D, et al. Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule. Exp Med. 2003 Aug 18; 198(4): 557-67. Epub 2003 Aug 11.
10．李州利，石炳毅，蔡明，等．血小板T细胞活化抗原1与移植肾急性排斥反应的相关性，肾脏与透析肾移植杂志，2003，12(3)：239-245．
11．叶志中，李富容，张丽君，等．系统性红斑狼疮患者T淋巴细胞PTA1表达的研究，中华风湿杂志，2002，6(4)：243-245．
12. Huang C, Jin B, Wang M, et al. Hemorrhagic fever with ernal syndrome: relationship between pathogenesis and cellular immunity. J Infect Dis, 1994; 169:868-870.
13. Nichoison JK, Heam TL,Gross GD,et al. 1997 advised guidelines for performing CD + T-cell determinations in persons infected with human immunodeficiency virus (HIV). MMWR Rcomm Rep, 1977, 46 (RR-2): 1-29.
14．五坚，胡绍文，王泊云，等．自身免疫性甲状腺病浸润T细胞活化与滤泡细胞DR抗原表达．中华医学杂志，1992；27：77-80．
15．贾卫，金伯泉，李琦，等．检测可溶性PTA1分子夹心ELISA方法的建立．细胞与分子免疫学杂志 2002，16(4)：282-284．
16. Jin B, Scott JL, Vadas MA, et al. TGF beta down-regulates TliSA1 expression and inhibits the differentiation of precursor lymphocytes into CTL and LAK cells. Immunology, 1989; 66:570-576.
17. TLiSA1 (PTA1) activation antigen implicated in T cell differentiation and platelet activation is a member of the immunoglobulin superfamily exhibiting distinctive regulation of expression. J Biol Chem, 1997; 272:21735-44.
18. Pantalco G, Mechanisms of CD8 + dysfunction in HIV infection. Fauci As, immunopathogenic mechanisms in human immunodefiencey virus (HIV) infection. Ann Tnter Med, 1991,114:678-693.
19. Tabata H, Hara M, Kitani A, et al. Hirose-T expression of TLiSAL on T cells from patients with rheumatoid arthritis and systemic lupus erythemtosus. Clin Immunol Immunopathol, 1989; 52:366-375.
20. Taylor JM, Fahey JL, Detels R, et al. CD4 percentage, CD4 number and CD4:CD8 ratio in HIV infection: which to choose and how to use. J Acquir Immune Defie Syndr. 1998, 2: 114-124.
21. Moretta L, Biossoni R, Bottino C et al. Human NK receptors. Immunnol Today, 2000, 21 (9) :420-422.
22. Diefenbach A, Raulet DH, Strategies for target cell recognition by natural killer cells. Immunological Review, 2001,181: 170-184.
23．张芸，欧阳为明，韩卫宁，等．CD226(PTA1)单抗诱导NK细胞克隆杀伤靶细胞的研究．中国免疫学杂志，2002，18(2)：79-81．
24. Chehimi J, Bandyopadhyay S, Prakash K, et al. In vitro infection of natural killer cells with different human immunodeficiency virus type 1 isolates. J Virol 1991 65(4): 1812-22.
25. Jewett A, Cavalcanti M, Giorgi J, et al. Concomitant killing in vitro of both gp120-coated CD4 + peripheral T lymphocytes and natural killer cells in the antibody-dependent cellular cytotoxicity (ADCC) system. J Immunol, 1997, 158(11): 5492-5500.
26. Oliva A, Kinter AL, Vaccarezza M, et al. Natural killer cells from human immunodeficiency virus (HIV)-infected individuals are an important source of CC-chemokines and suppress HIV-1 entry and replication in vitro. J Clin Invest, 1998, 102(1): 223-231.
27. Bruunsgaard H, Pederson C, Skinhoj P, et al. Clinical progression of HIV infection: role of NK cells. Scand J Immunol, 1997, 46(1): 91-95.
28. Ironson G, Balbin E, Solomon G, et al. Relative preservation of natural killer cell cytotoxicity and number in healthy AIDS patients with low CD4 cell counts. AIDS ,2001,15 (16):2065-2073.
29. Ullum H, Gotzsche PC, Victor J, et al. Defective natural immunity: an early manifestation of human immunodeficiency virus infection. J Exp Med, 1995, 182 (3): 789-799.
30. Mansour I, Doinel C, Rouger P, CD16 + NK cells decrease in all stages of HIV infection through a selective depletion of the CD16 + CD8 + CD3-subset. Immunol Today, 1990, 11:81-82.
31. Valentin A, Rosati M, Patenaude DJ, et al. Persistent HIV-1 infection of natural killer cells in patients receiving highly active antiretroviral therapy. Proc Natl Acad Sci U S A 2002, 14; ;99(10):7015-20.
32. Brown MG, Dokun AO, Heusel JW, et al. Vital involvement of a natural killer cell activation receptor in resistance to viral infection Science, 2001, 292 (5518): 934-937.
33. Andre P, Brunet C, Guia S, et al. Differential regulation of killer cell Ig-like receptors and CD94 lectin-like dimers on NK and T lymphocytes from HIV-1-infected individuals. Eur J Immunol, 1999, 29(4): 1076-1085.
34. Giavedoni LD, Valasquillo MC, Laura MP, et al. Cytokine expression, natural killer cell activation, and phenotypic changes in lymphoid cells from rhesus macaques during acute infection with pathogenic simian immunodeficiency virus. J Virology, 2000,1648-1657.
35. Parato KG, Kumer A, Badley AD, et al. Normalization of natural killer cell function and phenotype with effective anti-HIV therapy and the role of IL-10. AIDS,2002,16(9): 1251-1256.
36．宁双飞，贾卫，刘京梅，等．肾综合症出血热患者血清可溶性CD226／PTA1的检测和意义．细胞与分子免疫学杂志 2002，18(3)：283-284．
37．周武庆，宁双飞，贾卫，等．银屑病血清中可溶性血小板T细胞活化抗原1的检测．中华皮肤科杂志 2003，36(3)：159-160．
2. Leng Q, Borkow G, Weisman Z, et al. Immune activation correlates better than HIV plasma viral load with CD4 T-cell decline during HIV infection. J Acquir Immune Defic Syndr. 2001 Aug 1; 27(4): 389-97.
3. Hazenberg MD, Otto SA, van Benthem BH, et al. Persistent immune activation in HIV-1 infection is associated with progression to AIDS. AIDS. 2003 Sep 5; 17(13): 1881-8.
4. Savarino A, Bottarel F, Malavasi F, et al. Role of CD38 in HIV-1 infection: an epiphenomenon of T-cell activation or an active player in virus/host interactions? AIDS,2000; 14 (9): 1079-89.
5. Jia W, Lin XS, Zhu H, et al. Preparation and characterization of mabs against different epitopes of CD226 (PTA1). Hybridoma, 2000, 19 (5): 489-494.
6．田方，金伯泉、姜绍淳，等．一种新的免疫球蛋白超家族成员PTAI的细胞分布及表达研究．细胞与分子免疫学杂志，1996；12：53-57．
7. Sherrington PD, Scott JL, Jin B, et al. TliSA1 (PTA1) activation antigen implicated in T cell differentiation and platelet activation is a member of th immunoglobulin super family exhibiting distinctive regulation of expression. J Biol Chem 1997,272:21735-21744.
8. Chen L, Xie X, Zhang X, et al. The expression, regulation and adhesion function of a novel CD molecule, CD226, on human endothelial cells, Life Sci. 2003 Sep 19; 73 (18): 2373-82.
9. Bottino C, Castriconi R, Pende D, et al. Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule. Exp Med. 2003 Aug 18; 198(4): 557-67. Epub 2003 Aug 11.
10．李州利，石炳毅，蔡明，等．血小板T细胞活化抗原1与移植肾急性排斥反应的相关性，肾脏与透析肾移植杂志，2003，12(3)：239-245．
11．叶志中，李富容，张丽君，等．系统性红斑狼疮患者T淋巴细胞PTA1表达的研究，中华风湿杂志，2002，6(4)：243-245．
12. Huang C, Jin B, Wang M, et al. Hemorrhagic fever with ernal syndrome: relationship between pathogenesis and cellular immunity. J Infect Dis, 1994; 169:868-870.
13. Nichoison JK, Heam TL,Gross GD,et al. 1997 advised guidelines for performing CD + T-cell determinations in persons infected with human immunodeficiency virus (HIV). MMWR Rcomm Rep, 1977, 46 (RR-2): 1-29.
14．五坚，胡绍文，王泊云，等．自身免疫性甲状腺病浸润T细胞活化与滤泡细胞DR抗原表达．中华医学杂志，1992；27：77-80．
15．贾卫，金伯泉，李琦，等．检测可溶性PTA1分子夹心ELISA方法的建立．细胞与分子免疫学杂志 2002，16(4)：282-284．
16. Jin B, Scott JL, Vadas MA, et al. TGF beta down-regulates TliSA1 expression and inhibits the differentiation of precursor lymphocytes into CTL and LAK cells. Immunology, 1989; 66:570-576.
17. TLiSA1 (PTA1) activation antigen implicated in T cell differentiation and platelet activation is a member of the immunoglobulin superfamily exhibiting distinctive regulation of expression. J Biol Chem, 1997; 272:21735-44.
18. Pantalco G, Mechanisms of CD8 + dysfunction in HIV infection. Fauci As, immunopathogenic mechanisms in human immunodefiencey virus (HIV) infection. Ann Tnter Med, 1991,114:678-693.
19. Tabata H, Hara M, Kitani A, et al. Hirose-T expression of TLiSAL on T cells from patients with rheumatoid arthritis and systemic lupus erythemtosus. Clin Immunol Immunopathol, 1989; 52:366-375.
20. Taylor JM, Fahey JL, Detels R, et al. CD4 percentage, CD4 number and CD4:CD8 ratio in HIV infection: which to choose and how to use. J Acquir Immune Defie Syndr. 1998, 2: 114-124.
21. Moretta L, Biossoni R, Bottino C et al. Human NK receptors. Immunnol Today, 2000, 21 (9) :420-422.
22. Diefenbach A, Raulet DH, Strategies for target cell recognition by natural killer cells. Immunological Review, 2001,181: 170-184.
23．张芸，欧阳为明，韩卫宁，等．CD226(PTA1)单抗诱导NK细胞克隆杀伤靶细胞的研究．中国免疫学杂志，2002，18(2)：79-81．
24. Chehimi J, Bandyopadhyay S, Prakash K, et al. In vitro infection of natural killer cells with different human immunodeficiency virus type 1 isolates. J Virol 1991 65(4): 1812-22.
25. Jewett A, Cavalcanti M, Giorgi J, et al. Concomitant killing in vitro of both gp120-coated CD4 + peripheral T lymphocytes and natural killer cells in the antibody-dependent cellular cytotoxicity (ADCC) system. J Immunol, 1997, 158(11): 5492-5500.
26. Oliva A, Kinter AL, Vaccarezza M, et al. Natural killer cells from human immunodeficiency virus (HIV)-infected individuals are an important source of CC-chemokines and suppress HIV-1 entry and replication in vitro. J Clin Invest, 1998, 102(1): 223-231.
27. Bruunsgaard H, Pederson C, Skinhoj P, et al. Clinical progression of HIV infection: role of NK cells. Scand J Immunol, 1997, 46(1): 91-95.
28. Ironson G, Balbin E, Solomon G, et al. Relative preservation of natural killer cell cytotoxicity and number in healthy AIDS patients with low CD4 cell counts. AIDS ,2001,15 (16):2065-2073.
29. Ullum H, Gotzsche PC, Victor J, et al. Defective natural immunity: an early manifestation of human immunodeficiency virus infection. J Exp Med, 1995, 182 (3): 789-799.
30. Mansour I, Doinel C, Rouger P, CD16 + NK cells decrease in all stages of HIV infection through a selective depletion of the CD16 + CD8 + CD3-subset. Immunol Today, 1990, 11:81-82.
31. Valentin A, Rosati M, Patenaude DJ, et al. Persistent HIV-1 infection of natural killer cells in patients receiving highly active antiretroviral therapy. Proc Natl Acad Sci U S A 2002, 14; ;99(10):7015-20.
32. Brown MG, Dokun AO, Heusel JW, et al. Vital involvement of a natural killer cell activation receptor in resistance to viral infection Science, 2001, 292 (5518): 934-937.
33. Andre P, Brunet C, Guia S, et al. Differential regulation of killer cell Ig-like receptors and CD94 lectin-like dimers on NK and T lymphocytes from HIV-1-infected individuals. Eur J Immunol, 1999, 29(4): 1076-1085.
34. Giavedoni LD, Valasquillo MC, Laura MP, et al. Cytokine expression, natural killer cell activation, and phenotypic changes in lymphoid cells from rhesus macaques during acute infection with pathogenic simian immunodeficiency virus. J Virology, 2000,1648-1657.
35. Parato KG, Kumer A, Badley AD, et al. Normalization of natural killer cell function and phenotype with effective anti-HIV therapy and the role of IL-10. AIDS,2002,16(9): 1251-1256.
36．宁双飞，贾卫，刘京梅，等．肾综合症出血热患者血清可溶性CD226／PTA1的检测和意义．细胞与分子免疫学杂志 2002，18(3)：283-284．
37．周武庆，宁双飞，贾卫，等．银屑病血清中可溶性血小板T细胞活化抗原1的检测．中华皮肤科杂志 2003，36(3)：159-160．