中国HIV/AIDS患者NK细胞及其相关表面标志物研究
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摘要
目的
天然免疫系统是机体抵御外来病原体的第一道防线，在此基础上衍生出十分复杂的获得性免疫反应。近二十年来研究者们一直致力于获得性免疫对HIV感染的免疫应答研究，但到目前在免疫治疗和疫苗方面尚无突破性进展。近来研究显示：天然免疫系统在抵御HIV感染中占有非常重要的地位。自然杀伤细胞(Natural killer cells，NK)是天然免疫中的重要细胞，其表达CD56抗原不表达T细胞受体复合物TCR；自然杀伤T细胞(Natural killer T cell，NKT)是近来发现的NK细胞群的另一个组成部分，具有NK细胞表面标志物CD56同时亦表达TCR分子。NK和NKT细胞与HIV感染的关系目前尚无明确研究结果，本文首次研究了中国HIV／AIDS患者NK、NKT细胞及相关标志物的表达，初步探讨NK细胞与HIV感染的关系。
方法
1．研究对象
由辽宁省疾病预防与控制中心、吉林省疾病预防与控制中心以及中国医科大学艾滋病研究所艾滋病确认实验室经WB试验确认为阳性的HIV／AIDS患者。HIV抗体阴性健康对照，来自健康体检门诊和实验室工作人员，血液系统及肝肾功能检查结果正常，无免疫系统疾病史。
2．淋巴细胞绝对值和比值测定
将20ul IriTEST CD3FITC／CD56CD16PE／CD45PerCP或CD4FITC／CD8PE／CD3PerCP试剂加入绝对计数管中，经反向加样法加入50ul抗凝血，室温避光15min，加入免洗溶血素450ul，室温避光15min，FACS MULTISET软件检测并进行自动分析，计算细胞绝对值及相应比值等。以CD45~+
细胞为门，用CELLQUEST软件分析CD3‘/CD56CD16+/CD45+NKT细胞
数及相应比值等。
3.细胞表面染色
按照BD公司细胞表面染色说明书操作，应用FACSC亦bur流式细胞仪
检测10万个细胞，CELLQUEST软件分析NK细胞上表面标志物的表达。
4.核酸提取
应用全自动核酸分离纯化系统(MagNApureLC)和罗氏公司核酸提取
试剂盒，提取病毒翩A或人白细胞DNA。
5.病毒载量测定
采用RT一PCR方法，按照罗氏公司HIV病毒载量测定说明书进行操
作。
6.PCR扩增KIR3DLI和KIR3DSI基因
用引物KIR3 DLI(Fo二盯d)CGCTGTGGTGCCTCGA和KIR3DLI(Re-
verse)e盯GTGAAeeCeGACATG扩增KIR3DLI基因，得到197bp的片断。
用引物KIR3 DSI(Forward):AGCCTGCAGGGAACAGAAG和KIR3DSI(
Revers。)GccTGAcTGTGGTGTCG得到一300bp大小的片断。扩增条件为:
94℃3而nl个循环;94℃15 see，65℃15 see，72℃30 see，4个循环;94℃
15see，60℃15 see，72℃30 see，21个循环;94℃15 see，55℃1 min，72℃2而n，
5个循环，72℃7 min。取5林1 pCR产物经1.5%琼脂糖电泳，与DNA Maker
比对，判断结果。
7.HAART治疗方案
按照卫生部《艾滋病标准治疗方案》选取无明显肝、肾功能障碍的病
例，进行HAART治疗。口服默沙东公司的依非韦伦600毫克，每日一次，
硫酸苟地那韦1000毫克，每日三次。
结果
1.中国东北地区健康成年人NK和T淋巴细胞相关参考范围
NK细胞为121一916个/闪，NK%为6一35%。CD4‘T细胞为373-
1350个/闪，CDS+T细胞为240一1320个/闪，CD3‘T细胞为812一2790个/
闪，CD4/CDS比值为0.66一3.13。
2.HlwAIDs患者NK和NKT细胞与正常人的比较
比较HIWAIDS患者和正常对照NK、NKT、CD4+T、CDS+T细胞数及
相关比值。结果显示:HIWAIDS患者CD4十T细胞平均值为438 .4*290.2
个/林l，显著低于正常对照组CD4+T细胞(851.8土250.8个/林l)，p<0.01;
HIWAIDS患者CDS+T细胞平均值为1 1 11 .58土537.3个/闪，显著高于正
常对照组CDS‘T细胞(660.4士290.1个/林l)，p<0.01;HIV/AIDS患者NK
细胞平均值为278 .2*234 .5个/闪，显著低于正常对照组NK细胞(407 .0
土222.8个/林l)，p<0.01;HIV/AIDS患者NKT细胞平均值为100.5士65.0
个/闪，低于正常对照组NKT细胞(124.0，91.7个/闪)，p<0.05。Hlw
AIDS患者NK百分数(14.0士7.9%)亦显著低于正常对照组(18.8土8.
8%)。
3.HIWAIDS患者NK和NKT细胞与CD4、CDS细胞的相关性
HIWAIDS患者NK细胞的数量与CD4十T细胞数量成正比，r=0.289，
p<0.01;与CDS十T细胞数量不相关。NKT细胞的数量与CD4十T细胞数量
成正比，r=0.378，p<0.01;与CDS+T细胞数量成正比，r=0.340，p<0.
01。
4.长期不进展、慢性感染和艾滋病各组NK和NKT细胞变化的比较
根据CD4十T细胞数量和感染时间，不同HIWAIDS患者被分成三组:
CD4十T细胞数量大于或等于500个/闪、感染时间超过8年者为HIV长期
不进展(LTNP)组，CD4十T细胞数量介于500一200个/闪或等于200个/闪
者为HIV慢性感染组(Chronie infeetion)，CD4+T细胞数量小于200个/林l
者为艾滋病组。比较各组NK、NKT、CD4十T、CDS十T细胞数量及相关比值，
结果显示:
(l)慢性感染和艾滋病组NKT、NK细胞数量低于正常对照组(p<0.
05，p<0.Ol’)，慢性感染组NK细胞百分数显著低于正常对照组(p<0.01);
长期不进展组NKT、NK细胞数量与正常对照组无显著差别;但NK细胞百
分数显著低于正常对照组(p<0 .01);NKT细胞百分数在各组间无差别。
(2)艾滋病组CD4?
The innate immune system is the first line of defense against invading pathogens and from it the more sophisticated adaptive immune system was derived. Despite nearly two decades of research directed at inducing adaptive immunity or vaccine, there was no significant achievment has been made. On the basis of recent observations, it is suggested that the study on innate immune responses is very important in HIV infection. Natural killer cells ( NK) are the main cells in innate immune responses. The classical NK cells express CD56, lacking expression of TCRs, its phenotye is CD3- CD56 + ; Natural Killer T cells ( NKT) is a second set of NK cells expressing TCR molecules and NK cell surface markers CD56, its phenotype is CD3 + CD56 ". The responses and functions of NK cells and NKT cells in HIV infection are still not clear. In this work we have analysed the NK and NKT cells and their associated cell surface markers in HIV infection and investige the correlation between NK cells and HIV infection.
Material and Methods
1. Study population
Diagnosis of HIV - 1 infection was made on the basis of a positive anti -HIV ELISA ( Vironostika, Organon Tednika, The Netherlands ) and confirmed by a positive Western immunoblot ( Genelab Diagnostics, Singapore ). Treatment - naive subjects were asked to participate in the study. HIV - seronegative controls were randomly selected from healthy population with normal blood routine test, liver and renal function.
2. Determination of lymphocyte counts and corresponding ratio
Pipette 20ul of TriTEST CD4FITC/CD8PE/CD3PerCP or CD3FITC/ CD56CD16PE/CD45PerCP reagent and add 50ul anticoagulated whole blood into the bottom of the TruCOUNT Tube using reverse pipetting and incubate for 15 minutes in dark at room temperature (20 -25℃). Add 450ul 1X FACS Lysing Solution to the tube, incubate for 15minutes in the dark at room temperature and analyze the samples on the flow cytometer using FACS MULTISET software to acquire T lymphocyte counts, NK cell counts and corresponding ratios. Gating on CD45+ cells, acquire CD3 VCD56CD16VCD45 + NKT counts and corresponding ratios using CELLQUEST software.
3. Cell surface staining
According to the manual of cell surface staining kits of BD Company, 100, 000 cells were acquired and expression of cell surface markers on NK cells were analyzed using CELLQUEST software by FACSCalibur flow cytometer.
4. Isolation of nucleic acid
Viral RNA or human leucocyte DNA were isolated automatic nucleic acid extraction system (MagNApure LC) and nucleic acid extraction kits ( ROC).
5. Determination of viral load ( VL)
Viral load was performed by Roch viral load kits and preformed according to the kit instruction.
6. Amplification of KIR3DLland KIR3DS1 gene
Primers for KIR3DL1 gene: KIR3DL1 ( Forward) CGCTGTGGTGCCTCGA and KIR3DL1 (Reverse) GGTGTGAACCCCGACATG.
Primers for KIR3DSlgene: KIR3DS1 (Forward) AGCCTGCAGGGAA-CAGAAG and KIR3DS1 (Reverse) GCCTGACTGTGGTGTCG.
DNA extracted from peripheral blood mononuclear cells was used as templates. PCR cycling was carried out in a GeneAmp PCR 9600thermal cycler(PE Applied Biosystems) as follows; 1 cycle of 94℃ for 3min; 4 cycles of 94℃ 15 sec,65℃15 sec,72℃30 sec; 21 cycles of 94℃15sec,60℃ 15 sec,72℃30 sec; 5 cycles of 94℃. 15 sec,55℃1 min, 72℃ 2 min, 72℃7 min. 5ul amplified product was applied to 1.5% agarose gel electrophoresis and the results was determined comparing to DNA maker.
7. HAART regimen:
. Patients with no obvious liver and renal function abnormality were chosen according to the " standard therapy management" issued by the Ministry of Health and treated with antiviral drugs: Indinavir, a kind of proteinase inhibitor, taken orally 1000mg every 8 hours; Efavirenz, a kind of non - nucleoside reverse tran-scriptase inhibitor, taken orally 600mg daily.
Results
1. Reference range of NK cell and T lymphocyte counts of healthy adults in the northeast China
The reference range of NK cells is 121 -916 cell/ul,NK% is 6 -35% , CD4 is 373 - 1350 cell/ul,CD8 is 240 - 1320 ce
天然免疫系统是机体抵御外来病原体的第一道防线，在此基础上衍生出十分复杂的获得性免疫反应。近二十年来研究者们一直致力于获得性免疫对HIV感染的免疫应答研究，但到目前在免疫治疗和疫苗方面尚无突破性进展。近来研究显示：天然免疫系统在抵御HIV感染中占有非常重要的地位。自然杀伤细胞(Natural killer cells，NK)是天然免疫中的重要细胞，其表达CD56抗原不表达T细胞受体复合物TCR；自然杀伤T细胞(Natural killer T cell，NKT)是近来发现的NK细胞群的另一个组成部分，具有NK细胞表面标志物CD56同时亦表达TCR分子。NK和NKT细胞与HIV感染的关系目前尚无明确研究结果，本文首次研究了中国HIV／AIDS患者NK、NKT细胞及相关标志物的表达，初步探讨NK细胞与HIV感染的关系。
方法
1．研究对象
由辽宁省疾病预防与控制中心、吉林省疾病预防与控制中心以及中国医科大学艾滋病研究所艾滋病确认实验室经WB试验确认为阳性的HIV／AIDS患者。HIV抗体阴性健康对照，来自健康体检门诊和实验室工作人员，血液系统及肝肾功能检查结果正常，无免疫系统疾病史。
2．淋巴细胞绝对值和比值测定
将20ul IriTEST CD3FITC／CD56CD16PE／CD45PerCP或CD4FITC／CD8PE／CD3PerCP试剂加入绝对计数管中，经反向加样法加入50ul抗凝血，室温避光15min，加入免洗溶血素450ul，室温避光15min，FACS MULTISET软件检测并进行自动分析，计算细胞绝对值及相应比值等。以CD45~+
细胞为门，用CELLQUEST软件分析CD3‘/CD56CD16+/CD45+NKT细胞
数及相应比值等。
3.细胞表面染色
按照BD公司细胞表面染色说明书操作，应用FACSC亦bur流式细胞仪
检测10万个细胞，CELLQUEST软件分析NK细胞上表面标志物的表达。
4.核酸提取
应用全自动核酸分离纯化系统(MagNApureLC)和罗氏公司核酸提取
试剂盒，提取病毒翩A或人白细胞DNA。
5.病毒载量测定
采用RT一PCR方法，按照罗氏公司HIV病毒载量测定说明书进行操
作。
6.PCR扩增KIR3DLI和KIR3DSI基因
用引物KIR3 DLI(Fo二盯d)CGCTGTGGTGCCTCGA和KIR3DLI(Re-
verse)e盯GTGAAeeCeGACATG扩增KIR3DLI基因，得到197bp的片断。
用引物KIR3 DSI(Forward):AGCCTGCAGGGAACAGAAG和KIR3DSI(
Revers。)GccTGAcTGTGGTGTCG得到一300bp大小的片断。扩增条件为:
94℃3而nl个循环;94℃15 see，65℃15 see，72℃30 see，4个循环;94℃
15see，60℃15 see，72℃30 see，21个循环;94℃15 see，55℃1 min，72℃2而n，
5个循环，72℃7 min。取5林1 pCR产物经1.5%琼脂糖电泳，与DNA Maker
比对，判断结果。
7.HAART治疗方案
按照卫生部《艾滋病标准治疗方案》选取无明显肝、肾功能障碍的病
例，进行HAART治疗。口服默沙东公司的依非韦伦600毫克，每日一次，
硫酸苟地那韦1000毫克，每日三次。
结果
1.中国东北地区健康成年人NK和T淋巴细胞相关参考范围
NK细胞为121一916个/闪，NK%为6一35%。CD4‘T细胞为373-
1350个/闪，CDS+T细胞为240一1320个/闪，CD3‘T细胞为812一2790个/
闪，CD4/CDS比值为0.66一3.13。
2.HlwAIDs患者NK和NKT细胞与正常人的比较
比较HIWAIDS患者和正常对照NK、NKT、CD4+T、CDS+T细胞数及
相关比值。结果显示:HIWAIDS患者CD4十T细胞平均值为438 .4*290.2
个/林l，显著低于正常对照组CD4+T细胞(851.8土250.8个/林l)，p<0.01;
HIWAIDS患者CDS+T细胞平均值为1 1 11 .58土537.3个/闪，显著高于正
常对照组CDS‘T细胞(660.4士290.1个/林l)，p<0.01;HIV/AIDS患者NK
细胞平均值为278 .2*234 .5个/闪，显著低于正常对照组NK细胞(407 .0
土222.8个/林l)，p<0.01;HIV/AIDS患者NKT细胞平均值为100.5士65.0
个/闪，低于正常对照组NKT细胞(124.0，91.7个/闪)，p<0.05。Hlw
AIDS患者NK百分数(14.0士7.9%)亦显著低于正常对照组(18.8土8.
8%)。
3.HIWAIDS患者NK和NKT细胞与CD4、CDS细胞的相关性
HIWAIDS患者NK细胞的数量与CD4十T细胞数量成正比，r=0.289，
p<0.01;与CDS十T细胞数量不相关。NKT细胞的数量与CD4十T细胞数量
成正比，r=0.378，p<0.01;与CDS+T细胞数量成正比，r=0.340，p<0.
01。
4.长期不进展、慢性感染和艾滋病各组NK和NKT细胞变化的比较
根据CD4十T细胞数量和感染时间，不同HIWAIDS患者被分成三组:
CD4十T细胞数量大于或等于500个/闪、感染时间超过8年者为HIV长期
不进展(LTNP)组，CD4十T细胞数量介于500一200个/闪或等于200个/闪
者为HIV慢性感染组(Chronie infeetion)，CD4+T细胞数量小于200个/林l
者为艾滋病组。比较各组NK、NKT、CD4十T、CDS十T细胞数量及相关比值，
结果显示:
(l)慢性感染和艾滋病组NKT、NK细胞数量低于正常对照组(p<0.
05，p<0.Ol’)，慢性感染组NK细胞百分数显著低于正常对照组(p<0.01);
长期不进展组NKT、NK细胞数量与正常对照组无显著差别;但NK细胞百
分数显著低于正常对照组(p<0 .01);NKT细胞百分数在各组间无差别。
(2)艾滋病组CD4?
The innate immune system is the first line of defense against invading pathogens and from it the more sophisticated adaptive immune system was derived. Despite nearly two decades of research directed at inducing adaptive immunity or vaccine, there was no significant achievment has been made. On the basis of recent observations, it is suggested that the study on innate immune responses is very important in HIV infection. Natural killer cells ( NK) are the main cells in innate immune responses. The classical NK cells express CD56, lacking expression of TCRs, its phenotye is CD3- CD56 + ; Natural Killer T cells ( NKT) is a second set of NK cells expressing TCR molecules and NK cell surface markers CD56, its phenotype is CD3 + CD56 ". The responses and functions of NK cells and NKT cells in HIV infection are still not clear. In this work we have analysed the NK and NKT cells and their associated cell surface markers in HIV infection and investige the correlation between NK cells and HIV infection.
Material and Methods
1. Study population
Diagnosis of HIV - 1 infection was made on the basis of a positive anti -HIV ELISA ( Vironostika, Organon Tednika, The Netherlands ) and confirmed by a positive Western immunoblot ( Genelab Diagnostics, Singapore ). Treatment - naive subjects were asked to participate in the study. HIV - seronegative controls were randomly selected from healthy population with normal blood routine test, liver and renal function.
2. Determination of lymphocyte counts and corresponding ratio
Pipette 20ul of TriTEST CD4FITC/CD8PE/CD3PerCP or CD3FITC/ CD56CD16PE/CD45PerCP reagent and add 50ul anticoagulated whole blood into the bottom of the TruCOUNT Tube using reverse pipetting and incubate for 15 minutes in dark at room temperature (20 -25℃). Add 450ul 1X FACS Lysing Solution to the tube, incubate for 15minutes in the dark at room temperature and analyze the samples on the flow cytometer using FACS MULTISET software to acquire T lymphocyte counts, NK cell counts and corresponding ratios. Gating on CD45+ cells, acquire CD3 VCD56CD16VCD45 + NKT counts and corresponding ratios using CELLQUEST software.
3. Cell surface staining
According to the manual of cell surface staining kits of BD Company, 100, 000 cells were acquired and expression of cell surface markers on NK cells were analyzed using CELLQUEST software by FACSCalibur flow cytometer.
4. Isolation of nucleic acid
Viral RNA or human leucocyte DNA were isolated automatic nucleic acid extraction system (MagNApure LC) and nucleic acid extraction kits ( ROC).
5. Determination of viral load ( VL)
Viral load was performed by Roch viral load kits and preformed according to the kit instruction.
6. Amplification of KIR3DLland KIR3DS1 gene
Primers for KIR3DL1 gene: KIR3DL1 ( Forward) CGCTGTGGTGCCTCGA and KIR3DL1 (Reverse) GGTGTGAACCCCGACATG.
Primers for KIR3DSlgene: KIR3DS1 (Forward) AGCCTGCAGGGAA-CAGAAG and KIR3DS1 (Reverse) GCCTGACTGTGGTGTCG.
DNA extracted from peripheral blood mononuclear cells was used as templates. PCR cycling was carried out in a GeneAmp PCR 9600thermal cycler(PE Applied Biosystems) as follows; 1 cycle of 94℃ for 3min; 4 cycles of 94℃ 15 sec,65℃15 sec,72℃30 sec; 21 cycles of 94℃15sec,60℃ 15 sec,72℃30 sec; 5 cycles of 94℃. 15 sec,55℃1 min, 72℃ 2 min, 72℃7 min. 5ul amplified product was applied to 1.5% agarose gel electrophoresis and the results was determined comparing to DNA maker.
7. HAART regimen:
. Patients with no obvious liver and renal function abnormality were chosen according to the " standard therapy management" issued by the Ministry of Health and treated with antiviral drugs: Indinavir, a kind of proteinase inhibitor, taken orally 1000mg every 8 hours; Efavirenz, a kind of non - nucleoside reverse tran-scriptase inhibitor, taken orally 600mg daily.
Results
1. Reference range of NK cell and T lymphocyte counts of healthy adults in the northeast China
The reference range of NK cells is 121 -916 cell/ul,NK% is 6 -35% , CD4 is 373 - 1350 cell/ul,CD8 is 240 - 1320 ce
引文
1. Levy JA. The importance of the innate immune system in controlling HIV infection and disease. Trends Immunol 2001; 22(6):312-6
2. Diefenbach A. and Raulet D. Strategies.for target cell recognition by natural killer cells. Immunological Reviews 2001 ; 181 : 170 - 184
3. Miller J. The biology of natural killer cells in cancer, infection and pregnancy. Experimnetal hematology. 2001 ;29 : 1157 - 1168
4. Fortis C, Tasca S, et al. Natural killer cell function in HIV-1 infected patients. J Biol Regul Homeost Agents 2002 Jan-Mar; 16(1):30-2
5. Hinry M, Jackson Foundation, Rockville, et al. A comparative study of the impact of HIV infedtion on natural killer cell number and ruction in Thais and North Americans. AIDS Res Hum Retroviruses. 2000 Jul 20; 16 (11): 1061-6
6. Doherty DG, Norris S, Madrigal -Estebas L, et al. The human liver contains multiple populations of NK cells, T cells, and CD3~+ CD56~+ nutural T cells with distinct cytotoxic activities and Th1, Th2, and Th0 cytokine secretion patterns. J Immunol. 1999,163:2314 - 2321
7. Franitza S, Grabovsky V, Wald O, et al. Differential usage of VLA-4 and CXCR4 by CD3~+ CD56~+ NKT cells and CD56~+ CD16~+ NK cells regulates their interaction with endothelial cells. Eur. J. Immunol. 2004,34:1333-1341
8. Biron CA and Brossay L. NK cells and NKT cells in innate defense against viral infections. Current opinion in immunology. 2001;13:458-464
9. Unutmaz D. NKT cells and HIV infection. Microbes Infect. 2003; 5 (11): 1041-7.
10. van der Vliet HJ, von Blomberg BM, Hazenberg MD, et al. Selective decrease in circulating V alpha 24+ V beta 11+NKT cells during HIV type 1 infection, v J Immunol. 2002 Feb 1; 168 (3):1490-5.
11. Peruzzi M, Azzari C, Rossi ME, et al. Inhibition of natural killer cell cytotoxicity and interferon gamma production by the envelope protein of HIV and
prevention by vasoactive intestinal peptide. AIDS Res Hum Retroviruses, 2000;16(11) :1067 -73
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13. Zocchi MR, Rubartekki A, et al. HIV-1 Tat inhibits human natural killer cell function by blocking L- type calcium channels. J Immnnol 1998;161 (6):2938-43
14. Azzoni L, Papasavvas E, Chehimi J, et al. Sustained impairment of IFN-gamma secretion in suppressed HIV -infected patients despite mature NK cell recovery: evidence for a defective reconstitution of innate immunity. J Immunol 2002 ; 168 (11):5764-70
15. Galiani MD, Aguado E, Tarazona R, et al. Expression of killer inhibitory receptors on cytotoxic cells from HIV - 1 - infedted individuals. Clin Exp Immunol 1999 Mar; 115 (3):472-6
16. Tarazona R, Casado JG, et al et al. Selective depletion of CD56(dim) NK cell subsets and maintenance of CD56(bright) NK cell in treatment-nave HIV - 1 - seropositive individuals. J Clin Immunol; 2002;22 (3) : 176 - 83
17. Parato KG, Kumar 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):1251-6