心理應(yīng)激對(duì)正常人外周血T淋巴細(xì)胞體外活化表面分子表達(dá)的作用

1、    心理應(yīng)激對(duì)正常人外周血T淋巴細(xì)胞體外活化表面分子表達(dá)的作用        摘要目的：探討心理應(yīng)激增加對(duì)感染的易感性的細(xì)胞和分子免疫學(xué)機(jī)理。方法：采用雙熒光染色流式細(xì)胞分析法對(duì)20名健康大學(xué)生志愿者（男女各半）在應(yīng)激前后進(jìn)行外周血淋巴細(xì)胞免疫表型及T細(xì)胞體外絲裂原刺激早期活化抗原表達(dá)分析。連續(xù)一周期末考試被設(shè)定為心理應(yīng)激。結(jié)果：免疫表型分析顯示，應(yīng)激前后CD2、CD3、CD4、CD8、CD19、CD20、CD16、CD56等淋巴細(xì)胞的表面分子陽(yáng)性的細(xì)胞的百分比的差異無(wú)統(tǒng)

2、計(jì)學(xué)顯著性；與應(yīng)激前的結(jié)果相比，應(yīng)激后的T細(xì)胞在體外培養(yǎng)條件下多克隆刺激劑活化后CD69的表達(dá)明顯降低，植物血凝素（phytohemagglutinin,PHA）組CD69CD3/CD3的百分率由應(yīng)激前的28.1±4.1降低到應(yīng)激后的17.6±3.8，佛波醇酯（phorbol 12,13-dibutyrate,PDB）組CD69CD3/CD3的百分率由應(yīng)激前的80.7±6.8降至應(yīng)激后的65.8±7.9，而在沒有刺激劑作用的條件下，T細(xì)胞CD69表達(dá)率應(yīng)激前后的差別無(wú)顯著。結(jié)論：應(yīng)激對(duì)免疫系統(tǒng)的影響并不在于改變外周血淋巴細(xì)胞各亞群的比例的層面上；心理應(yīng)激

3、能降低健康人T細(xì)胞體外活化的反應(yīng)性，這可能與心理應(yīng)激個(gè)體對(duì)感染的易感性增加有關(guān)。主題詞心理學(xué)，醫(yī)學(xué)； T淋巴細(xì)胞； 抗原，CD69中分類號(hào)R392.9文獻(xiàn)標(biāo)識(shí)碼A文章編號(hào)1000-4718(2000)03-0229-04 Effects of psychological stress on in vitro expression of activated surface molecules on T cells of peripheral blood from healthy personsHUANG Bo-yan（Department of Biotechnology, Jinan Univ

4、ersity, Guangzhou 510632, China）SUN Hong, ZENG Jie-ming, WANG Xi, ZENG Yao-ying（Institute for Tissue Transplantation & Immunology, Jinan University）CHENG Yun-hua（First Affiliated Hospital of Jinan University Medical College)AbstractAIM: To study cellular and molecular mechanism involved in incre

5、asing susceptibility of infection in psychological stress persons. METHODS: Comparative studies were performed with double staining and flow cytometry analysis on immunophenotyping and in vitro expression of early activating surface molecule CD69 in response to mitogens on T cells from peripheral bl

6、ood of 20 healthy college student volunteers before and after psychological stress. A series of term final examinations was defined as psychological stress. RESULTS: Immunophenotyping analysis showed no statistically significant difference in the percentage of CD2, CD3, CD4, CD8, CD19, CD20, CD16 an

7、d CD56 positive lymphocyte populations before and after psychological stress. There was a statistically significant decrease in the in vitro expression of CD69 in response to polyclonal stimulators on the T cells from persons after psychological stress than those before psychological stress. The per

8、centage of CD69 expression (CD69CD3/CD3%) in response to PHA and PDB in the whole blood culture for 72 hours decreased respectively from 28.1±4.1 and 80.7±6.8 on the T cells obtained before psychological stress to 17.6±3.8 and 65.8±7.9 on those obtained after psychological stress

9、, while there was no statistically significant difference between the CD69 expression rates without stimulators on the T cells obtained before and after psychological stress. CONCLUSIONS: The effects of psychological stress to immune system is not on the level of changing proportions of the sub-popu

10、lations within peripheral blood lymphocytes. Psychological stress can decrease the activating response of T cells in healthy persons, which may be responsible for the increase of susceptibility to infection in the psychological stress persons.MeSHPsychological, medical; T-lymphocytes; Antigens, CD69

11、 有證據(jù)表明：腦、內(nèi)分泌系統(tǒng)和免疫系統(tǒng)共享同一套細(xì)胞因子、肽激素、神經(jīng)遞質(zhì)及相應(yīng)受體，因而這些分子就成為神經(jīng)內(nèi)分泌和免疫系統(tǒng)進(jìn)行雙向信息交流的化學(xué)語(yǔ)言1，2。雖然人們?cè)缫延^察到心理應(yīng)激（psychological stress）能增加個(gè)體對(duì)微生物感染的易感性3，但其細(xì)胞和分子機(jī)制尚未明了。本研究旨在探討心理應(yīng)激增加人體對(duì)感染的易感性的細(xì)胞和分子免疫學(xué)機(jī)制。材料和方法一、研究對(duì)象20名健康大學(xué)生志愿者參加此項(xiàng)研究，其中男、女學(xué)生各10名，年齡為1722歲。連續(xù)一周的期末考試被設(shè)定為心理應(yīng)激的刺激, 被調(diào)查者兼擔(dān)心所要面臨的考試難度較大， 有不及格的危險(xiǎn)?？荚囬_始前1周所抽取的靜脈血為心理應(yīng)激前樣

12、品，經(jīng)歷1周考試后所抽取的靜脈血為心理應(yīng)激后樣品。為排除不同時(shí)間免疫表型及體外活化表面分子表達(dá)的差別,我們?cè)趯W(xué)生準(zhǔn)備考試前3周已連續(xù)2次對(duì)他們進(jìn)行試驗(yàn)測(cè)定, 以排除其他心理應(yīng)激的可能性。其中受檢女學(xué)生兼排除月經(jīng)期，以排除激素水平變化的影響。二、單克隆抗體及試劑本研究所使用的CD69等單克隆抗體購(gòu)自法國(guó)IMMUNOTECH公司或美國(guó)Becton Dickinson 公司，PHA、PDB等多克隆刺激劑購(gòu)自美國(guó)Sigma公司，RPMI-1640、胎牛血清（fetal bovine serum, FBS）等細(xì)胞培養(yǎng)試劑購(gòu)自美國(guó)Gibco公司。三、實(shí)驗(yàn)方法熒光染色流式細(xì)胞分析法用于外周血淋巴細(xì)胞免疫表型

13、及T細(xì)胞體外絲裂原刺激早期活化抗原表達(dá)分析4，5。間接熒光標(biāo)記法被用于淋巴細(xì)胞免疫表型分析，程序步驟如下: 30L肝素抗凝靜脈血與10L第一單抗（鼠抗人）混合后室溫孵育30min，然后用紅細(xì)胞裂解液裂解紅細(xì)胞，離心獲取有核細(xì)胞，PBS洗滌后加入熒光標(biāo)記的羊抗鼠單抗（第二單抗），混合后室溫孵育30 min，PBS洗滌1次，用1多聚甲醛固定10 min后，用流式細(xì)胞儀進(jìn)行分析。間接-直接雙熒光標(biāo)記法被用于T細(xì)胞活化CD69表達(dá)的檢測(cè)，程序步驟與間接標(biāo)記法相似，只是在用多聚甲醛固定前插入以下步驟：加入熒光標(biāo)記的單抗后室溫孵育30 min并用PBS洗滌1次。T細(xì)胞活化采取體外全血培養(yǎng)多克隆活化劑刺激法

14、，抗凝全血用2倍完全RPMI-1640培養(yǎng)液稀釋，在CO2孵箱培養(yǎng)72 h。全部數(shù)據(jù)用FACStarplus流式細(xì)胞儀和LYSYS 軟件進(jìn)行單熒光或雙熒光參數(shù)獲取和分析。結(jié)果一、非考試同期自身對(duì)照及心理應(yīng)激前后免疫表型分析考試應(yīng)激前后及非考試同期自身對(duì)照CD2、CD3、CD4、CD8、CD19、CD20、CD16、CD56等淋巴細(xì)胞的表面分子陽(yáng)性的細(xì)胞的百分比的差異無(wú)顯著差別(見表1)。二、非考試同期自身對(duì)照及心理應(yīng)激前后T細(xì)胞體外活化分析與應(yīng)激前的結(jié)果相比，應(yīng)激后的T細(xì)胞在體外培養(yǎng)條件下多克隆刺激劑活化后CD69的表達(dá)明顯降低。而非考試同期自身對(duì)照CD69表達(dá)百分比的差異無(wú)顯著。體外全血培養(yǎng)

15、72 h，PHA組CD69?CD3/CD3的百分率由應(yīng)激前的28.1±4.1降低到應(yīng)激后的17.6±3.8，PDB組CD69CD3/CD3的百分率由應(yīng)激前的80.7±6.8降至應(yīng)激后的65.8±7.9，在沒有刺激劑作用的條件下，T細(xì)胞CD69表達(dá)率應(yīng)激前后的差別無(wú)顯著(見表2)。1顯示心理應(yīng)激前后T細(xì)胞體外活化后用流式細(xì)胞儀進(jìn)行分析時(shí)，各種情況下的實(shí)際記錄。表1非考試同期自身對(duì)照及心理應(yīng)激前后淋巴細(xì)胞免疫表型分析Tab 1Immunophenotyping of lymphocytes before and after psychological str

16、ess in normal persons (<"01.jpg (732 字節(jié))" src="/med/cano/201003/20100309195031927.jpg" 15 17>±s,n=20)T cellsB cellsNK cellsCD2CD3CD4CD8CD19CD23CD16CD56ControlStressFirst week74.9±8.366.1±6.642.7±6.727.7±6.96.5±2.72.3±1.413.7±5.814.5&#

17、177;5.2Secondweek73.8±7.965.2±7.048.0±7.225.7±7.17.2±2.13.1±1.814.2±4.615.6±6.4BeforePys.str75.0±8.064.1±7.544.6±6.826.8±6.55.6±1.92.5±1.712.8±4.813.8±4.8After Pys.str74.6±8.8*67.0±6.1*44.6±6.0*25.8±7

18、.2*7.0±1.8*1.8±2.3*14.8±6.2*12.7±5.4*     P0.05, vs control group; *P0.05, vs before psychological stress; Pys.str=psychological stress 表2非考試同期自身對(duì)照及心理應(yīng)激前后T細(xì)胞體外活化CD69表達(dá)的作用Tab 1Immunophenotyping of lymphocytes before and afterpsychological stress in normal perso

19、ns (<"01.jpg (732 字節(jié))" src="/med/cano/201003/20100309195031927.jpg" 15 17>±s,n=20)StimulatorsNonePHAPDBControlStressFirstweek1.5±0.826.7±3.981.0±7.1*Second week1.4±0.927.6±3.582.3±8.3*BeforePys.str1.6±1.128.1±4.180.7±6.8*Aft

20、erPys.str1.3±0.717.6±3.865.8±7.9*     P0.05, vs each other; *P0.05, vs each other; P0.01, vs PHA-stimulating group before psychological stress; *P0.01,vs PDB-stimulating group before psychological stress     Fig 1 Effect of psychological stress

21、on expression of CD69 by T cells during in vitro activation1心理應(yīng)激對(duì)T細(xì)胞體外活化CD69表達(dá)的作用討論人外周血淋巴細(xì)胞為非均一的細(xì)胞群，它包括T細(xì)胞、B細(xì)胞和NK細(xì)胞等細(xì)胞群，而T細(xì)胞又可分為T輔助細(xì)胞（T helper, Th）和細(xì)胞毒T細(xì)胞（cytolytic T lymphocytes, CTL）等若干亞群。在正常情況下，各群細(xì)胞按一定的比例形成某種正常布局，而在某些疾病狀態(tài)下這種正常布局會(huì)發(fā)生偏移。每群淋巴細(xì)胞都有特征性的細(xì)胞表面標(biāo)記分子，因而免疫表型分析的結(jié)果可揭示各群細(xì)胞的相對(duì)比例。免疫表型分析結(jié)果顯示：應(yīng)激前后總T細(xì)

22、胞、Th、CTL、B細(xì)胞和NK細(xì)胞的比例差異無(wú)顯著，這說(shuō)明應(yīng)激對(duì)免疫系統(tǒng)的影響不在于改變外周血淋巴細(xì)胞各亞群相對(duì)比例的層面上。T細(xì)胞在整個(gè)免疫應(yīng)答中起著關(guān)鍵性作用，而T細(xì)胞的活化效能是T細(xì)胞功能的反映。在某些疾病狀態(tài)下，T細(xì)胞的活化效能會(huì)發(fā)生改變。T細(xì)胞的活化效能可以通過在體外條件下對(duì)多克隆刺激劑的反應(yīng)來(lái)檢測(cè)。CD69是T細(xì)胞活化后表達(dá)的細(xì)胞表面分子68，用熒光抗體對(duì)CD3和CD69進(jìn)行雙色標(biāo)記后，再用流式細(xì)胞儀進(jìn)行分析可以準(zhǔn)確地檢測(cè)活化T細(xì)胞的相對(duì)數(shù)。我們的結(jié)果顯示，與應(yīng)激前的結(jié)果相比，應(yīng)激后的T細(xì)胞在體外培養(yǎng)條件下，經(jīng)多克隆刺激劑活化后，CD69的表達(dá)明顯降低，而在沒有刺激劑作用的條件下，

23、T細(xì)胞CD69的表達(dá)率在應(yīng)激前后的差別無(wú)顯著。我們的結(jié)果提示，心理應(yīng)激能降低健康人T細(xì)胞的活化反應(yīng)潛能，這可能與心理應(yīng)激個(gè)體感染的易感性增加有關(guān)?；痦?xiàng)目 國(guó)家自然科學(xué)基金資助(39670690)；廣東省自然科學(xué)基金資助(960196)黃柏炎（暨南大學(xué)生物工程學(xué)系， 廣東 廣州 510632）孫葒（暨南大學(xué)組織移植與免疫中心）曾潔銘（暨南大學(xué)組織移植與免疫中心）程云華（暨南大學(xué)醫(yī)學(xué)院第一附院）王曦（暨南大學(xué)組織移植與免疫中心）曾耀英（暨南大學(xué)組織移植與免疫中心）參考文獻(xiàn)1Blalock JE. The syntax of immune-neuroendocrine communication J. Immunol Today, 1994， 15(11): 504511.2Sternberg EM. Neural-immune interactions in health and diseas