紅細(xì)胞輸注對(duì)肝癌患者CD4+T輔助細(xì)胞免疫功能的影響

趙學(xué)濤，楊從容，任曉亮，李　明，解曉元，巨紅妹

(1.河北醫(yī)科大學(xué)第四醫(yī)院輸血科，河北 石家莊 050011；2.河北醫(yī)科大學(xué)第四醫(yī)院放療科，河北 石家莊 050011)

紅細(xì)胞輸注對(duì)肝癌患者CD4+T輔助細(xì)胞免疫功能的影響

趙學(xué)濤1，楊從容2，任曉亮1，李明1，解曉元1，巨紅妹1

(1.河北醫(yī)科大學(xué)第四醫(yī)院輸血科，河北 石家莊 050011；2.河北醫(yī)科大學(xué)第四醫(yī)院放療科，河北 石家莊 050011)

[摘要]目的探討輸入異體紅細(xì)胞對(duì)肝癌患者外周靜脈血中CD4+輔助性T淋巴細(xì)胞(helper T cells，Th)向Th1/Th2細(xì)胞亞群分化和相應(yīng)細(xì)胞因子分泌的影響。方法選取45例肝細(xì)胞癌患者，其中未輸血患者20例(未輸血組)，輸入過異體紅細(xì)胞患者25例(輸血組)。應(yīng)用流式細(xì)胞儀檢測(cè)2組患者外周血Th1(CD4+IFN-γ+)、Th2(CD4+IL-4+)的含量。采用酶聯(lián)免疫吸附測(cè)定法檢測(cè)2組患者外周血白細(xì)胞介素2(interleukin 2,IL-2)、干擾素γ(interferon-γ,INF-γ) 、IL-12(Th1型細(xì)胞因子)以及IL-4、IL-6、IL-10(Th2型細(xì)胞因子)的水平。結(jié)果輸血組Th1含量較未輸血組明顯降低(P<0.01)，而Th2含量較未輸血組則明顯升高(P<0.01)，但2組Th/CD4+T水平變化不明顯(P>0.05)。同時(shí)，輸血組IL-2、INF-γ和IL-12的水平明顯低于未輸血組(P<0.01)，IL-4、IL-6和IL-10的水平明顯高于未輸血組(P<0.01)。結(jié)論輸入異體紅細(xì)胞可導(dǎo)致肝癌患者體內(nèi)Th1/Th2平衡向Th2偏移，同時(shí)Th1亞群免疫反應(yīng)功能抑制，Th2亞群免疫反應(yīng)功能亢進(jìn)。臨床上應(yīng)盡量減少肝癌患者紅細(xì)胞輸注，以減輕對(duì)患者的免疫抑制。

[關(guān)鍵詞]肝腫瘤；紅細(xì)胞輸注；T淋巴細(xì)胞,輔助誘導(dǎo)

doi:10.3969/j.issn.1007-3205.2015.07.016

肝癌是嚴(yán)重威脅人類健康的主要疾病之一，也是癌癥死亡的主要原因[1]。肝癌患者常出現(xiàn)進(jìn)行性貧血，臨床多給予大量異體紅細(xì)胞輸注治療。然而大量回顧性研究表明異體輸血會(huì)降低機(jī)體免疫力,增加癌癥復(fù)發(fā)率和轉(zhuǎn)移率[2]，其具體機(jī)制尚不明確。輔助性T淋巴細(xì)胞(helper T cells，Th)的功能分化是機(jī)體免疫調(diào)節(jié)作用中的重要環(huán)節(jié)之一。其中Th1和Th2細(xì)胞亞群的平衡直接影響機(jī)體的免疫功能，并且與疾病狀態(tài)密切相關(guān)[3]。對(duì)于腫瘤患者，如Th1細(xì)胞活躍，則機(jī)體處于抵抗?fàn)顟B(tài)，反之則為易感狀態(tài)。異體紅細(xì)胞輸注對(duì)肝癌患者CD4+Th向Th1/Th2細(xì)胞亞群分化及其相應(yīng)細(xì)胞因子的影響目前尚未見報(bào)道，本研究擬針對(duì)這一環(huán)節(jié)進(jìn)行探討。報(bào)告如下。

1資料與方法

1.1一般資料選取2013年5月—2014年10月我院住院的肝癌患者45例，其中男性29例，女性16例，年齡46～74歲，中位年齡61歲。所有患者均經(jīng)病理確診為肝細(xì)胞癌。TNM臨床分期均為Ⅰ～Ⅱ期，無淋巴結(jié)轉(zhuǎn)移。全部患者未進(jìn)行任何抗腫瘤治療，且近3個(gè)月未使用免疫增強(qiáng)劑。45例患者中20例未進(jìn)行過輸血治療，為未輸血組，其中男性13例，女性7例，年齡46～69歲，平均(56.00±5.87)歲；25例曾輸注過異體紅細(xì)胞，平均輸血量2～4 U，為輸血組，其中男性16例，女性9例，年齡47～74歲，平均(58.00±6.94)歲。2組性別、年齡比較差異均無統(tǒng)計(jì)學(xué)意義(P>0.05)，具有可比性。

1.2Th1、Th2細(xì)胞檢測(cè)方法清晨空腹?fàn)顟B(tài)下留取患者外周靜脈血5 mL肝素鈉抗凝，1 h內(nèi)開始預(yù)處理。①刺激培養(yǎng): 取抗凝混勻全血1 mL+含10%胎牛血清的RPMI 1640液(Gibco公司)1 mL+BFA10 μL/ mL+PMA20 μg/L+離子霉1 mg/L吹打混勻，5%CO2、37 ℃孵育5 h。②胞外染色：將刺激培養(yǎng)的標(biāo)本分為3管，各500 μL用PBS洗1遍，1 200 r/min離心5 min，吸上清。每管加PEcy7-anti-CD4 抗體10 μL振蕩混勻，室溫避光15 min。③溶血及破膜：每管加10倍稀釋的FACS溶血素2 mL，室溫避光10 min，1 500 r/min離心5 min,棄上清。1%多聚甲醛固定細(xì)胞，各管加10倍稀釋的FACS通透劑0.5 mL室溫避光10 min，加PBS 2 mL,1 500 r/min離心5 min,棄上清。④胞內(nèi)染色：設(shè)1管為對(duì)照管，加同型對(duì)照FITC-anti-IgG2a/PE-anti-IgG1抗體(eBioscience公司)10 μL，余2管為試驗(yàn)管，分別加抗體FITC-anti-IFN-γ、PE-anti-IL-4(eBioscience公司)各10 μL混勻后室溫避光30 min。每管加1%多聚甲醛500 μL，流式細(xì)胞儀檢測(cè)。⑤結(jié)果判讀：在CellQuest軟件下獲取1萬個(gè)細(xì)胞，美國(guó)BD流式細(xì)胞儀測(cè)定淋巴細(xì)胞胞內(nèi)和胞膜各種熒光素的熒光強(qiáng)度。用PEcy7標(biāo)記的CD4單抗和側(cè)向角散射光雙參數(shù)圈出CD4陽性細(xì)胞群，空白對(duì)照和陰性對(duì)照消除自發(fā)熒光和非特異熒光，Dot2Plot點(diǎn)圖測(cè)Th1( CD4+IFN-γ+)、Th2(CD4+IL-4+)細(xì)胞百分比。

1.3細(xì)胞因子測(cè)定清晨空腹?fàn)顟B(tài)下留取患者外周靜脈血5 mL，不加抗凝劑，經(jīng)3 500 r/min離心5 min，取血清2～3 mL。采用酶聯(lián)免疫吸附測(cè)定(enzyme-linked immunosorbent assay，ELISA)法檢測(cè)細(xì)胞因子白細(xì)胞介素2(interleukin-2,IL-2)、干擾素γ(interferon-γ,IFN-γ)、IL-12(Th1型細(xì)胞因子)、IL-4、IL-6、IL-10(Th2型細(xì)胞因子)的含量，操作步驟按檢測(cè)說明書進(jìn)行。

2結(jié)果

2.12組Th1、Th2細(xì)胞水平比較輸血組Th1細(xì)胞水平與未輸血組比較明顯降低(P<0.01)，Th2水平與未輸血組比較顯著升高(P<0.01)，2組Th/CD4+T水平變化差異無統(tǒng)計(jì)學(xué)意義(P>0.05)，見表1。

表12組Th1、Th2細(xì)胞水平比較

Table 1Comparison of Th1 and Th2 levels between two groups

組別例數(shù)Th1(%)Th2(%)Th1/Th2Th/CD4+T未輸血組2025.35±2.4521.23±3.391.31±0.2747.35±3.61輸血組 2520.02±3.6426.32±5.640.87±0.2249.10±5.59t5.4403.4605.9300.990P0.0000.0010.0000.330

2.22組細(xì)胞因子含量比較輸血組肝癌患者血清IL-2、TNF-γ及IL-12含量明顯低于未輸血組，IL-4、IL-6和IL-10含量顯著高于未輸血組，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)，見表2。

表22組血清中細(xì)胞因子水平比較

Table 2Comparison of cytokine levels between two groups

組別例數(shù)IL-2(μg/L)IFN-γ(μg/L)IL-12(ng/L)IL-4(μg/L)IL-6(μg/L)IL-10(μg/L)未輸血組2017.19±4.2067.48±9.91145.51±11.551235.01±10.7214.78±2.9010.45±2.53輸血組 2512.44±4.9145.72±4.31128.64±12.29253.08±19.9710.58±1.5915.73±4.29t3.4309.8904.6303.5806.1624.870P0.0010.0000.0000.0010.0000.000

3討論

異體輸血在發(fā)揮臨床治療作用的同時(shí)，也會(huì)并發(fā)免疫相關(guān)的不良反應(yīng)，如術(shù)后感染率上升、惡性腫瘤復(fù)發(fā)率增加、慢性病毒感染的急性發(fā)作等[4-6]，這些輸血引起的一系列涉及免疫調(diào)節(jié)的反應(yīng)稱為輸血相關(guān)性免疫調(diào)節(jié)(transfusion-associated immunomodulation,TRIM)[7]。研究表明異體輸血不僅下調(diào)細(xì)胞免疫(包括T細(xì)胞和巨噬細(xì)胞功能)，而且下調(diào)體液免疫(包括自然殺傷細(xì)胞和巨噬細(xì)胞)，這些免疫調(diào)節(jié)作用可以部分解釋TRIM的發(fā)生[8-9]。

CD4+T細(xì)胞又稱Th，根據(jù)其分泌的細(xì)胞因子和介導(dǎo)的功能可再分為Th1細(xì)胞和Th2細(xì)胞。Th1細(xì)胞主要分泌IL-12、IL-2和IFN-γ，介導(dǎo)細(xì)胞免疫。而Th2細(xì)胞主要分泌IL-4、IL-5、IL-6、IL-10和IL-13等，介導(dǎo)體液免疫[10-12]。同時(shí)，Th1細(xì)胞分泌的IFN-γ可抑制Th2細(xì)胞，而IL-4能抑制Th1。因此，Th1和Th2細(xì)胞中一方占了優(yōu)勢(shì),另一方勢(shì)必受到抑制，從而使免疫應(yīng)答表現(xiàn)為以細(xì)胞免疫為主或以體液免疫為主的狀態(tài)。Th1和Th2可以互為抑制細(xì)胞，Th1和Th2細(xì)胞的平衡狀態(tài)直接影響到機(jī)體的免疫功能[13]。在腫瘤的發(fā)生發(fā)展中，細(xì)胞免疫是腫瘤免疫的主要組成部分，腫瘤患者Th1細(xì)胞及其分泌的細(xì)胞因子占優(yōu)勢(shì)促進(jìn)細(xì)胞免疫時(shí)，機(jī)體對(duì)腫瘤具有活躍的免疫力，但當(dāng)Th1/Th2細(xì)胞失衡，Th2細(xì)胞因子占優(yōu)勢(shì)抑制細(xì)胞免疫時(shí)，機(jī)體抗腫瘤免疫反應(yīng)受到抑制[14-16]。異體輸血導(dǎo)致的TRIM是否與Th1/Th2失衡有關(guān)呢？為此，本研究觀察了25例肝癌患者異體紅細(xì)胞輸注后對(duì)CD4+Th細(xì)胞免疫功能的影響，結(jié)果顯示，輸血組Th1水平較未輸血組明顯降低，Th2細(xì)胞水平明顯升高。表明異體紅細(xì)胞輸注提高了Th2反應(yīng)，而下調(diào)了Th1免疫反應(yīng)，導(dǎo)致Th1向Th2的偏移。本研究結(jié)果還顯示輸血組血清中IL-2、TNF-γ、IL-6含量明顯低于未輸血組，IL-4、IL-8、IL-10含量顯著高于未輸血組。表明異體紅細(xì)胞輸注導(dǎo)致Th1細(xì)胞因子分泌下降，而Th2細(xì)胞因子明顯升高，Th1/Th2向Th2偏移，Th2反應(yīng)占主導(dǎo)。從而證實(shí)異體輸血可能通過誘導(dǎo)Th1/Th2失衡而引發(fā)TRIM。

綜上所述，肝癌患者異體紅細(xì)胞輸注會(huì)改變CD4+Th細(xì)胞的免疫反應(yīng)，導(dǎo)致Th2反應(yīng)占主導(dǎo)，機(jī)體抗腫瘤反應(yīng)受到抑制，腫瘤轉(zhuǎn)移和復(fù)發(fā)的風(fēng)險(xiǎn)會(huì)有所增加。因此，臨床上應(yīng)盡量減少肝癌患者紅細(xì)胞輸注，以減輕對(duì)患者的免疫抑制。

[參考文獻(xiàn)]

[1]Siegel R,Ma J,Zou Z,et al.Cancer statistics，2014 [J].CA Cancer J Clin,2014,64(1):9-29.

[2]Cata JP,Wang H,Gottumukkala V,et al.Inflammatory response,immunosuppression,and cancer recurrence after perioperative blood transfusions[J].Br J Anaesth,2013,110 (5): 690-701.

[3]Talaat RM,Mohamed SF,Bassyouni IH,et al.Th1/Th2/Th17/Treg cytokine imbalance in systemic lupus erythematosus(SLE) patients:correlation with disease activity[J].Cytokine，2015 72(2):146-153.

[4]Blajchman MA.Transfusion immunomodulation or TRIM:what does it mean clinically? [J].Hematology,2005,10 (Suppl 1):208-214.

[5]Luan H,Ye F,Wu L,et al.Perioperative blood transfusion adversely affects prognosis after resection of lung cancer: a systematic review and a meta-analysis[J].BMC Surg，2014，14:34.

[6]Panagopoulos ND,Karakantza M,Koletsis E,et al.Influence of blood transfusions and preoperative anemia on long-term survival in patients operated for non-small cell lung cancer[J].Lung Cancer，2008，62(2):273-280.

[7]Aslam R,Speck ER,Kim M,et al.Transfusion-related immunomodulation by platelets is dependent on their expression of MHC Class Ⅰ molecules and is independent of white cells[J].Transfusion，2008，48(9):1778-1786.

[8]Federici AB,Vanelli C,Arrigoni L.Transfusion issues in cancer patients[J].Thromb Res,2012,219(Suppl 1):S60-65.

[9]Kaneko M,Sasaki S,Ishimaru K,et al.The impact of perioperative allogeneic blood transfusion on survival in elderly patients with colorectal cancer[J].Anticancer Res，2015，35(6):3553-3558.

[10]葉軍，羅軍敏.結(jié)核病T細(xì)胞亞群的研究進(jìn)展[J].免疫學(xué)雜志,2011,27(5):445-447.

[11]Wu TN,Lin KH,Chang YJ,et al.Avidity of CD1d-ligand-receptor ternary complex contributes to T-helper 1 (Th1) polarization and anticancer efficacy[J].Proc Natl Acad Sci USA,2011,108(42):17275-17280.

[12]Romagnani S.T-cell subsets (Th1 versus Th2)[J].Ann Allergy Asthma Immunol,2000,85(1):9-18.

[13]Ma J,Liu H,Wang X.Effect of ginseng polysaccharides and dendritic cells on the balance of Th1/Th2 T helper cells in patients with non-small cell lung cancer[J].J Tradit Chin Med，2014，34(6):641-645.

[14]Palomero-Rodríguez MA,Laporta-Bez Y,Snchez-Conde MP,et al.Inflammatory response,immunosuppression,and cancer recurrence after perioperative blood transfusions[J].Br J Anaesth,2014,112(3):576-577.

[15]Han YF,Zhao J,Ma LY,et al.Factors predicting occurrence and prognsis of hepatitis-B-virus-related hepatocellular carcinoma[J].World J Gastroenterol,2011,17(38):4258-4270.

[16]Lauerova L,Dusek L,Simickova M,et al.Malignant melanoma associates with Th1/Th2 imbalance that coincides with disease progression and immunotherapy response[J].Neoplasma,2002,49(3):159-166.

(本文編輯：趙麗潔)

[中圖分類號(hào)]R735.7

[文獻(xiàn)標(biāo)志碼]A

[文章編號(hào)]1007-3205(2015)07-0803-04

Influence of red blood cell transfusion on CD4+T-helper cells in liver cancer patients
ZHAO Xue-tao1,YANG Cong-rong2,REN Xiao-liang1,

LI Ming1,XIE Xiao-yuan1,JU Hong-mei1

(1.Department of Blood Transfusion,the Fourth Hospital of Hebei Medical University,Shijiazhuang

050011,China;2.Department of Radiation Oncology,the Fourth Hospital of Hebei Medical

University,Shijiazhuang 050011,China)

[Abstract]ObjectiveTo study the changes of Th1/Th2 subsets and cytokines of CD4+T-helper cells in peripheral blood of patients with hepatocellular carcinoma after blood transfusion of allogeneic red blood cells.MethodsThere were 45 patients with hepatocellular carcinoma，including 20 cases without blood transfusion(non-transfusion group) and 25 cases with blood transfusion of allogeneic red cells(transfusion group).The Th1 and Th2 in peripheral blood were examined in 45 patients by flow cytometry.Enzyme-linked immunosorbent assay was used to detect the level of serum interleukin-2(IL-2),IL-12,interferon-γ(INF-γ),IL-4,IL-6,and IL-10 in 45 patients.IL-2,INF-γ and IL-12 were used to represent cytokines of Th1 type,and IL-4,IL-6 and IL-10 to represent cytokines of Th2 type.ResultsThe level of Th1 in transfusion group was significantly lower than that in non-transfusion group(P<0.01),but the level of Th2 in transfusion group was significantly higher than that in non-transfusion group(P<0.01).The change of Th/CD4+T in two groups was not significant(P>0.05).The level of IL-2,TNF-γ and IL-12 in transfusion group were significantly lower than those in non-transfusion group(P<0.01),but the levels of IL-4,IL-6 and IL-10 were significantly higher in transfusion group than those in non-transfusion group(P<0.01).ConclusionBlood transfusion of allogeneic red cells induces a shift of the Th1/Th2 balance toward Th2 dominance.At the same time,subpopulation Th1 immune response was suppressed,Th2 subsets showed overactive immune response.Transfusion of allogeneic red blood cells should be avoided in liver cancer patients to reduce the immune suppression of blood transfusion.

[Key words]liver neoplasms；erythrocyte transfusion；T-lymphocytes,helper-inducer