于丹軍,樊 靜,門 帥,胡 月,姜 麗,曹麗艷,龔 珊

(1.秦皇島市第一醫(yī)院 檢驗(yàn)科，河北 秦皇島066000；2.秦皇島市第四醫(yī)院 檢驗(yàn)科，河北 秦皇島066000)

?

卵巢癌患者外周血調(diào)節(jié)性T細(xì)胞與TGF-β、IL-10檢測(cè)及其臨床意義

于丹軍1,樊 靜2,門 帥1,胡 月1,姜 麗1,曹麗艷1,龔 珊1

(1.秦皇島市第一醫(yī)院 檢驗(yàn)科，河北 秦皇島066000；2.秦皇島市第四醫(yī)院 檢驗(yàn)科，河北 秦皇島066000)

目的 探討卵巢癌患者手術(shù)前后及化療前后外周血CD4+CD25+調(diào)節(jié)性T細(xì)胞(Treg)比例，與TGF-β1、TGF-β2、IL-10含量變化的意義。方法 收集30例卵巢癌患者手術(shù)前后及化療后的外周靜脈血。采用流式細(xì)胞術(shù)檢測(cè)外周血Treg比例；用ELISA法檢測(cè)血清中細(xì)胞因子TGF-β1、TGF-β2、IL-10的含量，并以同期30例健康體檢者為對(duì)照組。結(jié)果 與健康對(duì)照組比較，卵巢癌患者CD4+CD25+CD127LOW占CD4+T淋巴細(xì)胞的比率、CD4+CD25+Foxp3+占CD4+T淋巴細(xì)胞比率及TGF-β1、IL-10含量明顯增高、TGF-β2明顯降低，差異有統(tǒng)計(jì)學(xué)意義(P< 0.01)，手術(shù)后及化療后外周血CD4+CD25+CD127LOW占CD4+T淋巴細(xì)胞的比率分別為[(1.610±0.465)%],[( 1.182±0.195)%],[( 1.033±0.210)%]；CD4+CD25+Foxp3+占CD4+T淋巴細(xì)胞比率分別為[(2.305±0.77)%],[(1.573±0.45)%],[(0.986±0.37)%]；細(xì)胞因子TGF-β1含量分別為(425.1±239.8) pg/ml,(287.5±239.8) pg/ml,(196.7±192.5) pg/ml；TGF-β2含量分別為(47.76±25.36) pg/ml，(32.22±17.81) pg/ml，(19.59 ±13.15) pg/ml；IL-10含量分別為(42.13±61.22) pg/ml,(17.34±11.21) pg/ml，(9.67±4.05) pg/ml，手術(shù)后及化療后卵巢癌患者CD4+CD25+CD127LOW占CD4+T淋巴細(xì)胞的比率、CD4+CD25+Foxp3+占CD4+T淋巴細(xì)胞比率及TGF-β1、TGF-β2、IL-10的含量較術(shù)前明顯減低(P<0.01)。結(jié)論 卵巢癌患者手術(shù)后及化療后外周血CD4+CD25+CD127LOW占CD4+T淋巴細(xì)胞的比率、CD4+CD25+Foxp3+占CD4+T淋巴細(xì)胞比率及血清中細(xì)胞因子TGF-β1、TGF-β2、IL-10水平明顯降低，動(dòng)態(tài)監(jiān)測(cè)它們的水平變化可作為評(píng)估卵巢癌患者免疫功能的可靠指標(biāo)，對(duì)其輔助診斷及療效觀察的評(píng)價(jià)具有重要臨床意義。

卵巢癌；調(diào)節(jié)性T細(xì)胞；Foxp3；細(xì)胞因子

(ChinJLabDiagn,2016,20:1655)

調(diào)節(jié)性T細(xì)胞(Treg)是一類具有免疫調(diào)節(jié)功能的抑制性淋巴細(xì)胞亞群，可以抑制免疫系統(tǒng)的抗腫瘤反應(yīng)，從而促進(jìn)腫瘤的發(fā)生、發(fā)展。同時(shí)它通過(guò)細(xì)胞接觸抑制機(jī)制釋放多種免疫抑制因子，如轉(zhuǎn)化生長(zhǎng)因子(TGF-β) 、IL-10等在誘導(dǎo)腫瘤的T細(xì)胞免疫耐受中發(fā)揮極其重要的作用[1,2]。轉(zhuǎn)錄因子叉頭蛋白P3(Foxp3)在Treg的發(fā)育過(guò)程中起關(guān)鍵作用，F(xiàn)oxp3的表達(dá)是Treg細(xì)胞的一個(gè)特異性免疫標(biāo)記[3]。本文通過(guò)檢測(cè)卵巢癌患者手術(shù)前后及化療后外周血中Treg細(xì)胞、Foxp3、TGF-β、IL-10的水平，為腫瘤的預(yù)防、診斷和治療提供可靠依據(jù)。

1 資料與方法

1.1 研究對(duì)象 選擇2014年8月至2015年8月在秦皇島第一醫(yī)院經(jīng)病理確診的卵巢惡性腫瘤患者(漿液性乳頭狀囊腺癌，中低度分化-低度分化)30例，年齡(42-75)歲，中位年齡55歲。選擇同時(shí)期來(lái)我院健康體檢者30名(健康對(duì)照組)，年齡(33-77)歲，中位年齡53歲。

1.2 標(biāo)本采集 卵巢癌患者在確診24 h內(nèi)未進(jìn)行放化療，采集靜脈血2 ml(卵巢癌組術(shù)前)；術(shù)后1個(gè)月采血2 ml(卵巢癌組術(shù)后)；化療4個(gè)周期后采血2 ml(卵巢癌組化療后)，健康體檢者采靜脈血2 ml(健康對(duì)照組)，EDTA-K2抗凝。

1.3 試劑與儀器 流式細(xì)胞儀使用用美國(guó)貝克曼公司提供的XL四色分析儀，鼠抗人FITC-CD4、PE-CD25、PC5-CD127和同型對(duì)照鼠抗人IgG均購(gòu)自美國(guó)貝克曼公司。Foxp3試劑盒購(gòu)自美國(guó)ebioscience公司，IL-10、TGF-β1、TGF-β2試劑盒均購(gòu)自Rapibio公司。

1.4 檢測(cè)方法 用流式細(xì)胞儀測(cè)定CD4+CD25+CD127low占CD4+T淋巴細(xì)胞比率，CD4+CD25+Foxp3+占CD4+T淋巴細(xì)胞比率，用酶標(biāo)儀檢測(cè)各組血清中TGF-β1、TGF-β2、IL-10含量，按照ELISA試劑盒說(shuō)明書嚴(yán)格操作。

2 結(jié)果

2.1 與對(duì)照組比較，卵巢癌患者外周血CD4+CD25+CD127LOW占CD4+T淋巴細(xì)胞的比率、CD4+CD25+Foxp3+占CD4+T淋巴細(xì)胞比率明顯增高、血清中TGF-β2含量明顯降低、IL-10含量明顯增高，差異有統(tǒng)計(jì)學(xué)意義(P<0.05),見表1。

表1 兩組外周血CD4+CD25+CD127LOW/CD4+及CD4+CD25+Foxp3+/CD4+TGF-β1、TGF-β2、IL-10的水平

2.2 卵巢癌患者手術(shù)后及化療后外周血CD4+CD25+CD127LOW占CD4+T淋巴細(xì)胞的比率、CD4+CD25+Foxp3+占CD4+T淋巴細(xì)胞比率、血清中TGF-β1、TGF-β2、IL-10含量明顯降低，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)，見表2。

表2 卵巢癌患者手術(shù)前后及化療后外周血CD4+CD25+CD127LOW/CD4+、CD4+CD25+Foxp3+/CD4+TGF-β1、TGF-β2、IL-10的水平

注：Pa：手術(shù)前后P值；Pb:化療后P值

3 討論

近年來(lái)研究顯示，卵巢癌、乳腺癌、肺癌、胃癌等惡性腫瘤患者的外周血和腫瘤局部微環(huán)境中Treg細(xì)胞比例明顯增高，很多研究也已經(jīng)證實(shí)在動(dòng)物腫瘤模型與腫瘤患者外周血以及局部腫瘤組織中Treg細(xì)胞均顯著升高[4-6]，因此大多數(shù)腫瘤患者抗腫瘤免疫功能低下與Treg細(xì)胞密切相關(guān)[7]。本研究中卵巢癌患者外周血CD4+CD25+CD127LOW占CD4+T淋巴細(xì)胞的比率、CD4+CD25+Foxp3+占CD4+T淋巴細(xì)胞比率與健康對(duì)照組相比明顯增高，從而說(shuō)明Treg細(xì)胞在腫瘤的發(fā)生、發(fā)展過(guò)程中起到抑制抗腫瘤免疫的作用。

Treg是具有顯著免疫抑制作用的CD4 T細(xì)胞亞群，具有免疫無(wú)能和免疫抑制兩大特性[8,9]，其對(duì)免疫系統(tǒng)功能具有抑制效應(yīng)，維持機(jī)體免疫穩(wěn)態(tài)，另外參與腫瘤的免疫調(diào)節(jié)功能，與腫瘤細(xì)胞的免疫逃逸與免疫耐受密切相關(guān)。Foxp3是Treg細(xì)胞生長(zhǎng)發(fā)育和發(fā)揮細(xì)胞抑制功能的重要因子，可以促進(jìn)腫瘤生長(zhǎng)抑制及機(jī)體抗腫瘤免疫應(yīng)答。本研究顯示卵巢癌患者Foxp3顯著升高，手術(shù)后CD4+CD25+CD127LOW占CD4+T淋巴細(xì)胞的比率、CD4+CD25+Foxp3+占CD4+T淋巴細(xì)胞比率均明顯降低，提示其與腫瘤負(fù)荷相關(guān)。原因可能是腫瘤細(xì)胞通過(guò)某種機(jī)制誘導(dǎo)CD4+CD25+細(xì)胞增殖與分化，而調(diào)節(jié)性T細(xì)胞的增加又有利于腫瘤的進(jìn)展。Treg可通過(guò)多種機(jī)制發(fā)揮其抑制免疫應(yīng)答的功能，包括表達(dá)抑制細(xì)胞表面蛋白如細(xì)胞毒T淋巴細(xì)胞相關(guān)抗原4(CTLA-4)，分泌抑制因子如腫瘤壞死因子-β(TGF-β) 和白細(xì)胞介素-10(IL-10)，導(dǎo)致代謝紊亂和直接的細(xì)胞溶解等。本研究卵巢癌患者，IL-10、TGF-β的水平異常升高，手術(shù)后其水平隨Treg水平減低而明顯降低。TGF-β可以通過(guò)抑制宿主免疫活性細(xì)胞，進(jìn)而促進(jìn)腫瘤的發(fā)生、發(fā)展，IL-10具有雙向免疫調(diào)節(jié)特性，它介導(dǎo)的免疫抑制機(jī)制是通過(guò)作用于不同的免疫細(xì)胞亞群，以多種方式發(fā)揮免疫抑制，造成機(jī)體抗腫瘤免疫逃逸。有研究證實(shí)Treg細(xì)胞在荷瘤鼠體內(nèi)，能通過(guò)TGF-β和IL-10的作用調(diào)節(jié)免疫抑制[10-12]。同時(shí)研究結(jié)果顯示，卵巢癌患者化療后外周血CD4+CD25+CD127LOW占CD4+T淋巴細(xì)胞的比率、CD4+CD25+Foxp3+占CD4+T淋巴細(xì)胞比率、TGF-β以及IL-10水平均明顯減低，可能與化療抑制腫瘤的發(fā)生發(fā)展有關(guān)。本文通過(guò)檢測(cè)Treg細(xì)胞、Foxp3、TGF-β以及IL-10水平，從而了解機(jī)體的免疫功能狀態(tài)，尤其對(duì)腫瘤患者診斷、治療及預(yù)后評(píng)估具有重要的理論和臨床意義。

[1]Huang X ,Zhu J,Yang Y .Protection against autoimmnunity in nonlymphopenie hosts by CD4+CD25+regulatory T cells is antigen-specific and requires IL-10 and TGF-beta [J].J Immunol,2005,175(7): 4283.

[2]Fantini M C,Becker C,Tubbe I,et al.Transforming growth factor beta induced FoxP3 regulatory T cells suppress Thl mediated experimental colitis[J].Gut,2006,55(5):671.

[3]Liao C，Xiao W，Zhu N，et al．Radiotherapy suppressed tumor-specific recruitment of regulator T cells via up-regulating microR-545 in Lewis lung carcinoma cells[J]．Int J Clin Exp Pathol，2015，8(3)：2535．

[4]Okita R，Saeki T，Takashima S，et al．CD4+CD25+regulatory T cells in the peripheral blood of patients with breast cancer and non-small cell lung cancer[J]．Oncol Rep，2005，14(5)：1269．

[5]Wolf AM，Wolf D，Steurer M，et al．Increase of regulatory T cells in the peripheral blood of cancer patients[J]．Clin Cancer Res，2003，9(2)：606．

[6]Hindley JP，F(xiàn)erreira C，Jones E，et al．Analysis of the Tcell receptor repertoires of tumor-infiltrating conventional and regulatory T cells reveals no evidence for conversion in carcinogen-induced tumors [J]．Cancer Res，2011，71(3)：736．

[7]Yang ZZ，Novak AJ，Stenson MJ，et al．Intratumoral CD4+CD25+regulatory T cell mediated suppression of infilitrating CD4+T cell in B-cell non-Hodgkin lymphoma[J]．Blood，2006,107(9):3639.

[8]Fontenot J D，Gavin M A，Rudensky A Y．Foxp3 programs the development and function of CD4+CD25+regulatory T cells[J]．Nat Immunol，2003，4(4)：330．

[9]Miyara M，Sakaguchi S．Natural regulatory T cells：mechanisms of suppression[J].Trends Mol Med，2007，13(3)：108．

[10]Liao C，Xiao W，Zhu N，et al．Radiotherapy suppressed tumor-specific recruitment of regulator T cells via up-regulating microR-545 in Lewis lung carcinoma cells[J]．Int J Clin Exp Pathol，2015，8(3)：2535．

[11]Ladoire S，Arnould L，Mignot G，et al．Presence of Foxp3 expression in tumor cells predicts better survival in HER2-overexpressing breast cancer patients treated with neoadjuvant chemotherapy[J]．Breast Cancer Res Treat，2011，125(1)：65．

[12]蘇 飛，王 欣，楊帥龍，等．經(jīng)肝動(dòng)脈化療栓塞術(shù)聯(lián)合無(wú)水乙醇瘤內(nèi)注射治療原發(fā)性肝癌的Meta分析[J]．中華實(shí)驗(yàn)外科雜志，2014，31(7)：1553.

Regulatory T cells in peripheral blood and IL-10、TGF-β detection and its clinical significance in patients with ovarian cancer

YUDan-jun,FANJing,MENShuang,etal.

(Clinicallaboratory,FirstHospitalofQinhuangdaoCity,Qinhuangdao066000,China)

Objective To explore the proportion of CD4+CD25+regulatory T cells in peripheral blood and the content variation of TGF-β1、TGF-β2、IL-10 in serum in patients with ovarian cancer before and after surgery and chemotherapy．Methods Collect 30 cases of patient’s peripheral venous blood with ovarian cancer before and after surgery and chemotherapy.Flow cytometry was used to detect the proportion of regulatory T cells; ELASA was used to detect content of TGF-β1、TGF-β2、IL-10 cytokines in serum,and 30 cases of healthy physical examination as the control group to the same period.Results Compared with healthy control group，the proportion of CD4+CD25+CD127LOWand CD4+CD25+Foxp3+regulatory T cells accounted for CD4+T lymphocytes and the content of TGF-β1、IL-10 in serum in patients with ovarian cancer was significantly higher ,TGF-β2 was significantly lower TGF-β2(P<0.01),the proportion of CD4+CD25+CD127LOWaccounted for CD4+T lymphocytes after surgery and chemotherapy was [(1.610±0.465)%],[( 1.182±0.195)%]and [( 1.033±0.210)%],respectively; The proportion of CD4+CD25+Foxp3+regulatory T cells accounted for CD4+T lymphocytes after surgery and chemotherapy was [(2.305±0.77)%],[(1.573±0.45)%]and [(0.986±0.37)%],respectively; The content of TGF-β1 cytokines after surgery and chemotherapy was (425.1±239.8) pg/ml,(287.5±239.8) pg/ml and (196.7±192.5) pg/ml,respectively; The content of TGF-β2 cytokines after surgery and chemotherapy was (47.76±25.36) pg/ml，(32.22±17.81) pg/ml and (19.59 ±13.15) pg/ml ,respectively; The content of IL-10 cytokines after surgery and chemotherapy was (42.13±61.22) pg/ml,(17.34±11.21) pg/ml and (9.67±4.05) pg/ml,respectively.The proportion of CD4+CD25+CD127LOWand CD4+CD25+Foxp3+regulatory T cells accounted for CD4+T lymphocytes and the content of TGF-β1、TGF-β2、IL-10 in serum in patients with ovarian cancer was significantly lower after surgery and chemotherapy (P< 0.01).Conclusion The proportion of CD4+CD25+CD127LOWand CD4+CD25+Foxp3+accounted for CD4+T lymphocytes and the level of TGF-β1、TGF-β2 and IL-10 in serum in patients with ovarian cancer were significantly decrease after surgery and chemotherapy,monitoring their changes in levels dynamically can be used as the reliability index of evaluating the immune function of patients with ovarian cancer,and has important clinical significance on the auxiliary diagnosis and evaluation of curative observation.

Ovarian cancer;Regulatory T cells;Foxp3;Cytokines

1007-4287(2016)10-1655-03

R737.31

A

2015-12-25)